Mesenchymal Stem Cells for the Treatment of Complex Anovaginal and Rectovaginal Fistulas in Crohn's Disease: A Systematic Review

Keywords

stem cell - fistula - Crohn's Disease

Introduction

Crohn's disease (CD) is a chronic inflammation of unknown origin that affects the entire gastrointestinal system. One of the frequent and unexpected complications of the disease is the fistula, which consists of an abnormal, tunnel-like connection between the intestine and nearby epithelial surfaces.[1] [2] It is one of the inflammatory bowel diseases (IBDs) that primarily affects individuals aged 15 to 35. There are specific sex differences in Crohn's disease, with a predominance of women in the USA and Europe. This differs from Asian countries, where men are more likely to develop the disease.[3]

Unlike other inflammatory diseases, IBDs are not easily treatable.[4] Therapeutic strategies in Crohn's disease vary according to the phenotype of the condition. Thiopurines are essential in maintenance therapy to reduce steroid use, while anti-tumor necrosis factor agents play a crucial role, especially in the fistulizing form of the disease.[5] The disease causes symptoms such as pain, diarrhea, fever, and other discomforts. In addition to severely affecting the lower part of the small intestine, Crohn's disease can affect various regions of the digestive tract, including the large intestine, stomach, esophagus, and even the oral cavity.[4]

Individuals with Crohn's disease may present various perianal lesions associated with the condition, such as anal tags, lesions in the anal canal, including fissures, ulcers, and strictures, as well as fistulas, perianal abscesses, and cancer. The presence of perianal fistulas is an indication of an unfavorable long-term prognosis in patients with Crohn's disease.[6]

Fistulas affecting the vagina, such as rectovaginal fistulas (RVFs) and anovaginal fistulas (AVFs), include the passage of gas and/or stool through the vagina.[7] Female patients also report the presence of purulent discharge, dyspareunia, pain, and sensitivity in the perineal area, as well as recurrent urinary infections.[8]

Although an infection in an anal gland can lead to a low rectovaginal fistula in inflammatory bowel diseases, higher fistulas are likely a result of the penetrating nature of the inflammation characteristic of Crohn's disease.[9]

Perianal fistula associated with Crohn's disease (pfCD) is often resistant to treatment, resulting in continuous relapses and generating significant economic impact, as well as compromising patients' quality of life.[10] Current treatment options include biological medications, combination therapy with thiopurines, drug monitoring, and rigorous follow-up. Surgery is essential to drain abscesses before immunosuppression and to place setons. Once inflammation is controlled, definitive interventions such as fistulotomies and ligation of the fistula tract may be considered.[11]

In this context, mesenchymal stromal cells (MSCs) emerge as a potential therapy due to their ability to differentiate into various cell types and their immunomodulatory function. These cells can aid in tissue regeneration, promoting healing and reducing inflammation, which may be crucial for the treatment and healing of perianal fistulas.[10] The effective use of mesenchymal stem cells (MSCs) in the treatment of a refractory rectovaginal fistula in patients with Crohn's disease was first reported in 2003.[8]

Although MSCs can be easily obtained from adipose tissue, liposuction is an invasive surgical procedure. The umbilical cord (UC) emerges as a promising alternative to adipose tissue. Compared to MSCs derived from adult adipose tissue, UC-MSCs can be cultured for longer periods and have greater proliferation capacity.[10]

This work aims to review the efficacy and safety of the use of mesenchymal stem cells in the treatment of complex anovaginal and rectovaginal fistulas associated with Crohn's disease. Considering the resistance of these fistulas to conventional therapies, research into regenerative properties may offer new solutions.

Methods

This systematic review was performed and reported the following recommendations of the Cochrane Collaboration Handbook for Systematic Review of Interventions and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) Statement guidelines.[12] [13]

The development method consisted of searching for articles on the research platforms PubMed, Cochrane Library, Embase and Web of Science to answer the following research questions: How effective and safe are mesenchymal stem cells in treating complex anovaginal and rectovaginal fistulas associated with Crohn's disease? Based on the PICO strategy (P: Patients with complex anovaginal and rectovaginal fistulas associated with Crohn's disease; I: Mesenchymal stem cells; C: Conventional medical and/or surgical treatments; O: Efficacy and safety) the following search terms (MeSH) were used: stem cell, stromal cells, stem cells, fistula, fistulas, fistulizing, inflammatory bowel disease and Crohn's disease, as well as this free term: Sampling reflex which, associated with Boolean operators (OR and AND), structured the search strategy.

Research Bases

For this review, the broad search platforms PubMed, Cochrane Library, Embase and Web of Science on 08/10/2024 were searched.

Research Strategy

Search strategy: ("stem cell" OR "precursor cell" OR "precursor cells" OR "progenitor cell" OR "progenitor cells" OR "stromal cell" OR "stromal cells" OR "stem cells") AND ("fistula" OR "fistulas" OR "fistulizing") AND ("inflammatory bowel disease" OR "Crohn's disease" OR "crohn disease" OR "regional enteritis" OR "ileocolitis" OR "granulomatous colitis" OR "granulomatous enteritis" OR IBD)

Selection

For the selection, the Rayyan® Platform (https://www. rayyan.ai/) was used. The articles resulting from the search strategies were added to the platform and five collaborators were invited for the blind selection based on the reading of abstracts and titles. Conflict review was allowed for all employees. Once the conflicts were resolved, the articles included in the blind selection were read in full. Then, the final inclusion of articles was made.

Quality Assessment

Quality assessment of Randomized Controlled Trials (RCTs) was performed using version 2 of the Cochrane risk-of-bias tool for randomized trials (RoB 2).[10] In this tool, studies are scored as high, low, or unclear risk of bias in 5 domains: selection, performance, detection, attrition, and reporting biases. For observational studies, the Risk Of Bias In Non-Randomized Studies Of Interventions (ROBINS-I)[11] was used. This tool was employed to assess the risk of biases in observational studies, providing a comprehensive framework for evaluating the methodological quality of non-randomized studies of interventions. Two authors independently performed the quality assessment of the included studies (P.H.G.G. & A.J.S.O.B.), and any conflict was resolved by a third author (K.M.).

Results

Study Selection

A total of 1197 records were identified from PubMed (n = 291), Embase (n = 463), Cochrane (n = 76), and Web of Science (n = 367). After removing 555 duplicates, 642 records remained for title and abstract screening, we excluded papers that did not fulfill all of the eligibility criteria (n = 633), yielding a total of 9 articles for full-text screening. Reasons for deferral among the papers included: studies with population overlap (n = 2). A detailed step-by-step recording of the selection stages and process can be found in [Figure 1]. Ultimately, 7 articles were included in this sistemática review.

Fig. 1 PRISMA flow diagram.

Baseline Characteristics of the Included Studies and Patients

We included seven studies in this review: two phase IB/IIA trials,[8] [14] two phase I trial,[15] [16] one compassionate use program [Herreros 2019], one case series,[17] and one pilot study.[18] These studies collectively involved 126 patients treated for Crohn's disease-related fistulas, including perianal, rectovaginal, and anovaginal fistulas. The median follow-up ranged from 6 to 52 weeks.

The stem cell therapies used across the studies included expanded allogeneic adipose-derived mesenchymal stem cells,[16] [17] [19] autologous adipose-derived stem cells,[15] and bone marrow-derived mesenchymal stem cells.[8] [14] Administration methods generally involved direct intralesional injections, with some studies incorporating repeat dosing at three months.[8] [14] The majority of studies reported no serious adverse events related to stem cell therapies.

Patient characteristics indicated a predominance of females, particularly in the studies of rectovaginal fistulas.[14] [16] [17] The compassionate use program[19] included a more diverse patient cohort, with both Crohn's-associated and cryptoglandular fistulas. Ages across studies ranged from 18 to 72 years, with patients having a history of multiple unsuccessful surgical interventions or failures with biological treatments. Disease activity indices and fistula-related scores showed significant improvements in most studies, with complete healing rates ranging from 25% to 60%, depending on the type of fistula and stem cell source.

Data on race and ethnicity were not consistently reported across the studies. Nevertheless, the inclusion of patients from various treatment protocols and clinical centers underscores the potential applicability of these therapies in different settings. [Table 1] presents a comprehensive summary of the baseline characteristics of the included studies.

Table 1 Baseline Characteristics of Included Studies

Study	Design	Country	Total sample size	IBD*	Age (range), years	Type of fistula	Origin of mesenchymal cells	Complications	Improvements in symptons
GALA M. GODOY-BREWER, 2023[18]	Prospective multicenter clinical trial	USA	15	5 (not specified)	median 46.7 (31–72)	Rectovaginal fistula	Umbilical cord–derived tissue grafts	1 perirectal abscess 56 weeks after	12/18 repairs
LIGHTNER, 2020 [15]	Phase I safety trial	USA	5	5 Crohn's disease	median 49 (38–53)	single-tract rectovaginal fistula	Matrix-Delivered Autologous Mesenchymal Stem Cell	NR	5 ( all these fistula tracts had significantly diminished in diameter size by >50%)
LIGHTNER, 2023 [8]	Phase IB/IIA, randomised, control trial	USA	22 (16 experimental/ 6 control).	22 Crohn's disease of the pouch	median 41.2	Rectovaginal fistula: 3 (50.0%) - control / 7 (43.8%) - experimental // perianal: 3 (50%) - control / 9 (56%) - experimental	Bone Marrow-derived, Mesenchymal Stem Cells	Perianal pain 3 [50.0%] control ,11 [68.8%] experimental. Bleeding resolved on its own) 1 [16.7%] control, 2 [12.5%] experimental. Perianal abscess 1 [6.25%] experimental	Experimental 0 Decreased symptoms
LIGHTNER, 2024 [14]	Phase IB/IIA randomized control trial was performed in a 3:1, single-blinded study.	USA	19 (15 experimental/ 4 control)	10 Crohn's disease	median 42.6	Rectovaginal	Ex vivo allogeneic expanded bone marrow-derivade, mesenchymal stem cells	Perianal pain 2 (28.6) experimental Bleeding, 1 (14.3) experimental	100% had improvement in symptoms, with 92% reporting a decrease in drainage by 50% or more.
GARCIA-ARRANZ, 2016 [16]	Phase I–IIa Clinical Trial	Spain	10	10 Chron's disease	mean 35	Rectovaginal fistula	Allogeneic Expanded-Adipose Derived Stem Cells	NR	3/5 patients who completed the trial
HERREROS, 2019 [19]	Observational study	Spain	7	NR	mean 45 (24-69)	Rectovaginal fistula	Autologous adipose-derived, or allogenic adipose-derived stem cells	NR	6/7
NIKOLIC, 2020 [17]	Prospective study	Austria	4	4 Crohn's disease	median 52 (32-66)	Rectovaginal fistula	Allogeneic expanded adipose-derived stem cells	NR	1/4

Abbreviations: *Inflamatory Bowel diases (IBD).

Risk of Bias Assessment

Studies were generally assessed as having a low risk of bias across most domains, except for confounding, which presented serious concerns in all studies ([Fig. 2A]). One study showed a moderate risk in the measurement of outcomes due to methodological imprecision. In contrast, the studies in [Figure 2B] were consistently rated as having a low risk of bias across all evaluated domains, ensuring the high reliability of their findings.

Fig. 2 Quality assessment of the included studies using the RoB2 tool for the randomized studies (A) and ROBINS-I for non-randomized studies (B).

Discussion

Anovaginal fistulas (AVFs) associated with Crohn's Disease (CD) develop as a consequence of localized inflammation. These fistulas significantly impair patients' quality of life by causing fecal incontinence through the fistula, passage of stool into the vagina, leading to infections, pain and sexual dysfunction, as well.[7] [20] [21]

The treatment of AVFs varies depending on their size and complexity. However, many patients ultimately require surgical intervention, which has become the most common therapeutic approach. Among surgical options, anorectal flaps and interposition grafts are frequently employed, but their success rates range from 33% to 100%. This variability leaves up to half of the patients dissatisfied, with diminished quality of life, high recurrence rates, and persistent fecal incontinence.[20] [22] [23]

Hotouras et al. demonstrated that gracilis muscle interposition achieved a median healing rate of 100% over a 21-month follow-up period. However, patient monitoring was inconsistent, and limited data on complications were provided.[24] Similarly, Egal et al. reported a success rate of 63.6% for AVF closure using mucosal advancement flaps combined with isolated muscle plication. These findings highlight the variability in outcomes and the need for further research to optimize treatment strategies.[25]

In this context, the use of expanded ASCs has emerged as a promising approach for tissue regeneration and repair. These cells possess anti-inflammatory properties and differentiation potential, which may significantly contribute to the treatment of anovaginal fistulas.[26] A notable example is the study by Garcia-Olmo et al., which reported a healing rate of up to 75%, with no associated risk of incontinence or adverse effects, underscoring the therapeutic potential of ASCs.[27]

The first successful use of MSCs to treat refractory RVF in Crohn's disease was reported in 2003, sparking numerous clinical trials to evaluate their safety and efficacy. Despite this, only 17 RVF cases have been included across all trials, with most studies being single-center. Notably, Lightner et al conducted the first randomized phase 2 trial using MSCs to treat rectovaginal CD, highlighting their high safety profile and promising results in perianal disease.[8]

Building on these advancements, additional studies, although still in their early phases, have shown promising results in the treatment of AVFs associated with Crohn's disease. These advances highlight the therapeutic potential of mesenchymal stem cells in promoting fistula healing, despite methodological limitations and data heterogeneity. Therefore, the aim of this review is to analyze the key aspects of AVF treatment and recovery using MSCs, discussing findings in light of previous studies to identify gaps and potential advancements.[8] [14]

However, a critical analysis of the studies reveals significant variability in the MSC doses used, as there is currently no widely accepted protocol for the ideal dosage in fistula treatment. This variability, combined with anatomical differences in the fistulas, such as location and complexity, and the heterogeneity of studied populations— often lacking a clear diagnosis of CD or inflammatory bowel disease (IBD) — complicates standardization efforts and directly impacts closure rates.

Additionally, patients with AVFs frequently undergo multiple procedures, both surgical and non-surgical, and most have a history of prior medication use, which may influence their response to MSC therapy.[28] Another challenge lies in the absence of standardized definitions or uniform criteria to assess clinical or radiological remission, making direct comparisons between studies difficult and hindering the formulation of generalizable conclusions about the efficacy of this therapeutic approach.

A meta-analysis by Cao et al., which included anovaginal, perianal, and transsphincteric fistulas in patients with Crohn's disease (CD) and inflammatory bowel disease (IBD), demonstrated superior efficacy and a lower incidence of adverse events with stem cell (SC) therapy compared to conventional treatments. Specifically, SC therapy achieved a fistula healing rate of 58% to 62% and a clinical remission rate of 62.52% in patients with Crohn's fistulas, aligning with their prior findings. These results underscore the potential of SC therapy as a promising approach for managing complex fistulas, particularly in the context of CD.[1]

Similarly, Herreros et al. evaluated patients with complex perianal fistulas and found that among seven individuals with rectovaginal fistulas (RVFs), only three were related to CD. Despite the absence of complete healing at six months, six patients exhibited a 50% closure of external, vaginal, and rectal openings, as well as a 50% reduction in external drainage. Notably, only one of these seven RVF cases was treated with allogeneic stem cells (ASCs), while the others received stromal vascular fraction therapy. Their previous study reported a 60% closure rate, potentially attributed to the clinical complexity of selected cases and the administration of up to two doses of ASCs. The lower dosage and the use of autologous cells in some instances may also account for the observed differences in outcomes.[19]

Garcia-Arranz et al. explored fistula re-epithelialization using expanded mesenchymal stem cells (e-ASCs). Patients who did not achieve complete re-epithelialization after the first dose were rescued with a second application at double the initial dose, achieving 50% recovery in these cases. The study demonstrated the safety of donor-derived e-ASCs based on the observed side-effect profile. Furthermore, cytokine level analysis revealed no significant differences before and after treatment, indicating good tolerability of the administered cells. These findings underscore the therapeutic potential of e-ASCs, even in cases resistant to the initial treatment.[16]

More recently, Lightner et al 2020 evaluated the use of a fistula plug coated with autologous adipose tissue-derived mesenchymal stem cells in five patients with CD and anovaginal fistulas (AVFs). After six months of follow-up, no cases of complete clinical remission, defined as total cessation of drainage and absence of a rectovaginal tract under anesthesia, or radiographic remission, characterized by the absence of a rectovaginal tract on magnetic resonance imaging, were observed. However, three patients (60%) reported clinical healing, evidenced by total cessation of drainage. These results suggest that the technique may provide partial symptom relief, though the absence of complete remission highlights the need for optimization of the therapeutic approach.[15]

One factor that may have contributed to the more favorable clinical outcomes compared to the study by Nikolic et al. was the intestinal diversion performed in all patients at the time of surgery. These observations suggest that intestinal diversion may play a critical role in fistula healing and the effectiveness of MSC therapy.[17]

This finding has also been observed in other studies, where patients had previously undergone ostomies,[8] [14] [18] but it was not directly associated with better local recovery. Vaginal involvement in cases of CD-related fistula leads to worse outcomes according to Scott et al and a higher rate of ileostomy diversion or proctectomy.[29] [30] [31]

This might be due to the altered quality of rectal and perianal tissue and the fact that this is a poorly vascularized and thin area—as well as the poor healing that characterizes CD and relates to some of its underlying pathogenic mechanisms.[22]

Additionally, the study by Nikolic et al. found that of the four patients evaluated, only one achieved complete healing. This patient was the only one without previous surgeries related to CD, possibly indicating a less severe disease profile. These findings suggest that disease severity and the number of prior procedures may impact the effectiveness of MSC-based therapies. In this context, mesenchymal stem cells may prove more effective as a first-line treatment for anovaginal fistulas rather than as a therapy for recurrent or advanced cases.

Conversely, in the study by Duraes et al., among 18 treated patients, 13 had undergone previous repair attempts, and of these, 8 demonstrated symptom improvement or complete healing. These findings indicate that while factors such as disease severity and prior procedures influence outcomes, the efficacy of MSCs can still be significant in patients with a history of multiple treatments. Nonetheless, studies with larger patient cohorts are urgently needed to evaluate the impact of these factors and guide the development of tailored treatment strategies for different patient profiles.

Abbreviations

CD: Crohn's disease

IBDs: Inflammatory bowel diseases

AVFs: Anovaginal fistulas

RVFs: Rectovaginal fistulas

pfCD: Perianal fistula associated with Crohn's disease

MSCs: Mesenchymal stromal cells

UC: Umbilical cord

SC: Stem cell

e-ASCs: Expanded mesenchymal stem cells

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