A randomized trial study on the effect of amniotic membrane graft on wound healing process after anal fistulotomy O A m a G H a b c a A R A A K A H W P h 2 B j coloproctol (rio j). 2 0 1 7;3 7(3):187–192 w w w . j c o l . o r g . b r Journal of Coloproctology riginal Article randomized trial study on the effect of amniotic embrane graft on wound healing process after nal fistulotomy hahramani Leila a, Pirayeh Saeideh a, Khazraei Hajar a, Bagher pour Ali a, osseini Seyed Vahid a, Noorafshan Ali b, Safarpour Ali Reza c,∗, Mousavi Laleh a Shiraz University of Medical Sciences, Colorectal Research Center, Shiraz, Iran Shiraz University of Medical Sciences, Anatomy Department, Stereology Research Center, Shiraz, Iran Shiraz University of Medical Sciences, Gasteroentrohepatology Research Center, Shiraz, Iran r t i c l e i n f o rticle history: eceived 19 December 2016 ccepted 27 March 2017 vailable online 15 May 2017 eywords: nal fistula uman amniotic membrane ound healing ost-operative complication a b s t r a c t Objective: Human amniotic membrane (HAM) used as a wound coverage for more than a century. The aim of this study is to evaluate the efficacy of amniotic membrane on wound healing and reduce post-operative complication. Study design: Randomized clinical trial study. Place and duration of study: Surgery Department, Shahid Faghihi Hospital, Shiraz, in the period of between Sep. 2014 and Nov. 2015. Methodology: 73 patients with anal fistula were divided into two groups. The patients suffered from simple perianal fistula (low type) without any past medical history. Fistulotomy were performed for all of them and in interventional group HAM were applied as biologic dressing. Their wound healing improvement was evaluated post-operative in two groups. Results: From 73 patients participated in the study, 36 patients were in control group and 37 patients were in intervention group. According to the analysis of images taken from the wound, the rate of wound healing was 67.39% in intervention group and 54.51% in control group (p < 0.001). Discharge, pain, itching and stool incontinency was lower in intervention group. Analysis of pathology samples taken from the wound showed no differences between two groups. Conclusion: HAM application could lead to improvement of wound healing and reduced post- operative complications. In conclusion, HAM may act as a biologic dressing in the patients with anal fistula. © 2017 Sociedade Brasileira de Coloproctologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). ∗ Corresponding author. E-mail: colorectal2@sums.ac.ir (S.A. Reza). ttp://dx.doi.org/10.1016/j.jcol.2017.03.006 237-9363/© 2017 Sociedade Brasileira de Coloproctologia. Published by Elsevier Editora Ltda. This is an open access article under the CC Y-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). dx.doi.org/10.1016/j.jcol.2017.03.006 http://www.jcol.org.br http://crossmark.crossref.org/dialog/?doi=10.1016/j.jcol.2017.03.006&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ mailto:colorectal2@sums.ac.ir dx.doi.org/10.1016/j.jcol.2017.03.006 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ 188 j coloproctol (rio j). 2 0 1 7;3 7(3):187–192 Ensaio clínico randomizado sobre o efeito do enxerto de membrana amniótica sobre o processo de cicatrização após fistulotomia anal Palavras-chave: Fístula anal Membrana amniótica humana Cicatrização da ferida Complicação pós-operatória r e s u m o Objetivo: Membrana amniótica humana (MAH) tem sido usada para cobrir feridas por mais de um século. O objetivo deste estudo é avaliar a eficácia da membrana amniótica na cicatrização de feridas e reduzir complicações pós-operatórias. Desenho do estudo: Ensaio clínico randomizado. Local e duração do estudo: Departamento de Cirurgia, Shahid Faghihi Hospital, Shiraz, Irã, entre setembro de 2014 a novembro de 2015. Método: 73 pacientes com fístula anal foram divididos em dois grupos. Os pacientes sofriam de fístula perianal simples (tipo baixo) sem histórico médico prévio. A fistulotomia foi real- izada em todos eles e no grupo intervenção, MAH foi aplicada como curativo biológico. A melhora da cicatrização foi avaliada no período pós-operatório em dois grupos. Resultados: De 73 pacientes que participaram do estudo, 36 pacientes eram do grupo controle e 37 pacientes do grupo intervenção. De acordo com a análise das imagens da ferida, a taxa de cicatrização foi 67,39% no grupo intervenção e 54,51% no grupo controle (p < 0,001). Secreção, dor, prurido e incontinência fecal foi menor no grupo intervenção. A análise das amostras patológicas retiradas da ferida não mostrou diferenças entre os dois grupos. Conclusão: A aplicação de MAH pode levar à melhoria da cicatrização de feridas e reduzir as complicações pós-operatórias. Em conclusão, a MAH pode atuar como um curativo biológico nos pacientes com fístula anal. © 2017 Sociedade Brasileira de Coloproctologia. Publicado por Elsevier Editora Ltda. Este é um artigo Open Access sob uma licença CC BY-NC-ND (http://creativecommons.org/ Introduction Fistula-in-ano disease usually exists after anorectal infection. There are many treatment options for management of anal fis- tulas with minimum chance of incontinence and recurrence. Surgical managements have to eliminate the septic foci and any associated epithelized tract to avoid recurrence and pre- serve the anal sphincter function. All of the options have different success rates. Fistulotomy used in the underlying sphincter tissue and is recommended for low fistulas with reported success rates varying from 29% to 53%. Success rates with plug have been comparable or inferior to the advancement flap (48–62%). The flap should consist of the part of the internal sphincter and mucosa with a broad base of blood supply and should be sutured without tension. The success rate can be raised by removing the underlying infected anal gland and curetting the rest of the tract.1 Setonisa less invasive approach with minimal damage to the sphincter. However the discomfort caused to the patient during the long time required for wound healing is the main disadvantage of this approach. However, a cutting seton can have better (up to 99%) success rate, it can cause severe dis- comfort to the patient and also, can have 18–25% incidence of incontinence. Draining seton can have 20–40% persistent fistula rate, but with a low incidence of incontinence.2 In 2006, ligation of inter sphincteric fistula tract (L.I.F.T.) introduced by Rojanasakul for the first time as a total sphinc- ter saving procedure.3 Healing rate after 6–7 weeks is usually ranging from 68% to 83%. Video assisted anal fistula treat- ment (VAAFT) described by Prof. Meinero, that is done with licenses/by-nc-nd/4.0/). the rigid endoscope and the tract is cauterized, curetted and the internal opening is stapled.4 Cochrane database have described that no major difference was seen between the various techniques used if recurrence rates are concerned.5 Thus there is no single method that is perfect and physician has to choose the surgery depending on his/her experience, the type of fistula and the other local conditions. Many post-operative complications are because of dysfunc- tion of wound healing. Vascularity of anal canal is important but the main reason is infection and lack of scare recovery due to scare situation and humid dressing. So, complications like pain, itching, discharge and recurrence occurred. Human amniotic membrane (HAM) is the inner layer of the fetal membranes and has bio-compatibility, easy availability, elasticity and stability and it has been used as an alternative biomaterial for research in many surgeries and wound-healing procedures. Amniotic membrane has been used in different organs for example, many surgeons evaluated the efficacy of HAM as a biologic dressing in burn wounds or in corneal epithelium reconstruction with transplantation of epithelial cells on a lyophilized amniotic membrane (LAM) or in gastroin- testinal tract surgeries.6,7 Many studies assessed the efficacy of HAM as a biologic dressing in skin ulcers reported better outcomes in comparison to some other methods. Moreover, in a few studies, HAM has been evaluated in GI tract of animal models and the results showed accelerating wound healing process.8 Uludag et al. used HAM patch in colon anastomo- sis in rats and reported that using HAM decreases dehiscence rate, intra-abdominal abscesses, anastomotic leakage, adhe- sion formation and intestinal obstruction.9 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ j coloproctol (rio j). 2 0 1 7;3 7(3):187–192 189 Assessed for eligibility (n=80) clinically diagnosed anal fistula and fistulotomy done Excluded (n=7) leaved study Group A: HAM applied (n=37) Randomization Group B: without HAM (n=36) Follow-up 28 days Analysis: Itching, gas and stool incontinence, discharge, pain Follow-up 28 days Analysis: Itching, gas and stool incontinence, discharge, pain dia d t u t fi M T t p u M 2 c a g o i fi l a e m > Fig. 1 – Flow However, HAM has been put into practice for less than a ecade and more studies are needed for better evaluation and he probable long-term adverse effects of HAM should be eval- ated in further studies. The aim of this study was to evaluate he HAM effect on wound healing acceleration in the post stulotomy procedure. ethodology he study has been designed as a randomized clinical trial o evaluate efficacy of HAM in healing of fistula in-Ano. 73 atients with clinical diagnosis of fistula in-Ano were eval- ated in Shahid Faghihi Hospital of Shiraz University of edical Sciences between September 2014 and November 015. All patients suffered low type fistula in-Ano that was onfirmed by colorectal surgeon with physical examination nd anoscopy. The patient were randomly allocated into two roups; fistulotomy with marsupialization and HAM applying n wound in group A and fistulotomy with marsupialization n group B as control group (standard procedure for low type stula). The inclusion criteria were as follows: clinical diagnosis of ow type fistula (sphincter involvement <30%), age 18–65 years, nd American society of anesthesiologists class I or II. The xclusion criteria included the following; 1) immune compro- ised patients such as T.B, AIDS or DM received steroid drugs 20 mg/day; 2) inflammatory bowel disease; 3) past medical gram RCT. history of previous anal surgeries; 4) history of gas or stool incontinence; 5) allergy to egg; 6) refuse to participation in this study; 7) BMI > 30; 8) fistula with abscess; 9) high type fistula (sphincter involvement >30%); 10) previous pelvic radiation; 11) perianal dermatitis. Written informed consent form was filled by all the patients that participated in the study before surgery. Ran- domization was done by block randomization permuted total patients was 80 that 73 participants allocated in each group (we lost 7 patients in follow-up). The patients in the two groups received prophylactic dose of metronidazole just before anes- thesia and two doses post-operatively at 8 and 16 h. All patients operated in prone position, after anoscopy and iden- tification of fistula tract and internal, external orifice of fistula. Fistulotomy was done in eligible participants. Performing fis- tulatomy and curettage in group A, then HAM applied on the wound of fistula. HAM was fixed on the side of wound by monocryl 4/0 in four points the same as marsupialization. Then, digital photography was taken from 10 cm distance. Finally surgical dressing was applied. In group B, after fistulo- tomy and curettage, marsupialization was done in four points of the side wall with monocryl 4/0 and dressing the same as group A. Digital photography was taken also from 10 cm dis- tance as well. Normal diet was start after 1 day and dressing was removed. Then, the patients discharge in second days, if they did not have any complications such as unpredictable pain, abnormal discharge and cellulites. Both groups were operated by colorectal surgeon. Another colorectal surgeon 190 j coloproctol (rio j). 2 0 1 7;3 7(3):187–192 Table 1 – Demographic data from patients under simple surgery and HAM with surgery (percent). Group Male Female Mean age ± SD Simple surgery 31 (86.1%) 5 (13.9%) 39.94 ± 10.77 Surgery with HAM 20 (54.1%) 17 (45.9%) 37.32 ± 10.27 Table 2 – Discharge, itching, pain, incontinence parameters. Parameter p-value Odds ratio 95% confidence interval Lower Upper Discharge 0.000 2.29 1.53 3.42 Itching 0.000 4.82 2.65 8.78 1 – Pain <0.0001 Fecal incontinence 0.007 visited the patients 3, 7, 14, 21 and 28th days post operation that he was blinded to allocation of the two groups. Follow-up data form was complete with attention to sign and symptom of the patients (Fig. 1). Itching, gas and stool incontinence, dis- charge, pain scoring was determined by using VAS system. In second visit (2 weeks post operation), digital photograph was taken again in the same distance. Tissue biopsy was taken of 10 patients in both groups randomly. Therefore, primary out- come in this study was wound healing acceleration by HAM that was evaluated subjectively and objectively. Secondary outcome was infection and abscess formation. Question forms evaluated wound healing and infection subjectively and digi- tal photograph (digital image: stereolith), tissue biopsy taken helped us for objective evaluation (10 participants in each group). This study was approved by the ethics committee of Shiraz University of Medical Sciences and was registered with the Iranian Clinical Trial Register (IRCT: 201310219936N6). Statistics Statistical analysis was performed by SPSS software (version 16) and also SAS (for categorical repeated measurement). In descriptive analysis quantitative variables were revealed by mean ± SD, and qualitative variables were showed by fre- quency and percent. Qualitative variables were pain, discharge and itching. Quantitative variable was percent of scar recovery. They were measured during times after surgery. Repeated measurement analysis (RMA) was done for evaluation of sig- nificant changes in the outcome variables. Qualitative RMA and quantitative RMA were performed by SPSS and SAS soft wares respectively. Generalized estimating equation (GEE) was the method for discharge assay. Two samples t-test, �2 test and fisher exacted test also were uses in appropriate comparisons. p-value greater than 0.05 was considered significant. Results 80 patients evaluated and 7 of patients leave the study, 36 of them had simple fistulatomy (5 female and 31 male) and 37 of patients had fistulatomy with HAM graft (17 female and 20 male). In this study, mean age of patients with simple fistu- latomy was 39.9 years and mean age of patients with HAM was 37.3 years with no significant difference (Table 1). .61 1.34 1.93 −0.72 −0.11 In this study, variables like sex, age, history of fissure before surgery, time and type of surgery and their effects on discharge assayed (Table 2). Time and discharge had significant differ- ence (p = 0.003), that means increase of time decrease chance of discharge (OR = 0.96). Also, surgery with HAM in comparison with simple fistulatomy decrease chance of discharge more than two times (OR = 2.29). Sex, age and fissure did not have significant difference and showed that two groups were equal as sex and age. Itching and fissure before surgery, type of surgery and time had significant difference. GEE results in itching showed sig- nificant difference in time (p = 0.004) and by increase of time, chance of lack of itching increased (OR = 1.04). There was sig- nificant difference between two groups for itching (p < 0.05) and chance of lack of itching in group 1 was more than 4 times of group zero (OR = 4.82). Fissure in clinical exam before surgery affects itching significantly (p < 0.05). Chance of lack of itching in patients with fissure was lower than patients with- out fissure (OR = 0.17). Sex and age did not show any difference on itching (p = 0.421, p = 0.07), respectively. For Analysis the data for Pain SAS software used and GEE marginal modeling method showed that time significantly affected (p < 0.05) and by spending more time chance of lack of pain increased (OR = 2.14). Two groups demonstrated signif- icant difference in pain (p < 0.05) and chance of lack of pain in group zero was less than group 1 (OR = 0.47). So, surgery with HAM suggested as better surgery in comparison to another surgery. Sex and age did not show any difference on pain. Percent of healing According to the photographic data in day of surgery and 14 days after that, percent of scare recovery obtained by digi- tal image analysis. Mean ± SD of percent of recovery in group without HAM was 54.51 ± 4.86 and in group that used HAM graft was 67.39 ± 4.69. The difference between two groups was significant (p < 0.0001) that means use of HAM increased rate of scare recovery. Fecal incontinence parameter Lack of fecal incontinency in interventional group was signif- icantly less than control group (p-value = 0.007). Wexner score was used for incontinency evaluation. 2 0 1 7 P M a p D U f a s t fi s c d c k a e e p e t g r i e d o h b t e b l i l r c t s t p f s r a fi s u T q r 1 j coloproctol (rio j). athology ann–Whitney test used for comparison between two groups nd there was no significant difference between them as athologic data (p-value = 0.76). iscussion sually 70.7% of fistulas were healed in at least 1 year of ollow-up. Fistula-in-ano is a challenging condition to man- ge despite the technological advances and it is not a gold tandard treatment algorithm for it. Low transsphincteric fis- ulas are treated by fistulotomy successfully while complex stulas are managed by advancement flap repair, cutting eton, partial fistulotomy, stem cell injection, fibrin or dermal ollagen glue injection, plug, VAAFT, LIFT, and FiLaC, but evi- ence on healing, recurrence, and safety of these options is not larified completely. A study on anal fistula is needed to define ind of fistula (low, high, transsphincteric, intersphincteric) nd outcome measures (healing time, incontinence). Human amniotic membrane (HAM) has bio-compatibility, asy availability, elasticity and stability that researchers have ncouraged to consider it as a biologic dressing and appro- riate bio-prosthesis for more than 100 years. Many surgeons xamined the efficacy of HAM as a biologic dressing in their reatment methods such as burn wounds treatment or in astrointestinal tract surgeries and desirable outcomes were eported.6 Amnion cells synthesize peptides of the innate mmunity system, like as beta-defensins, elastase-inhibitors, lafin, lactoferrin, or IL-1-RA.HAM had antimicrobial effect ue to these immune factors. Also, HAM synthesizes numer- us growth factors such as epithelial growth factor (EGF), uman growth factor (HGF), keratinocyte growth factor (KGF), asic fibroblast growth factor (bFGF), and tissue growth fac- ors (TGF-alpha, TGF-beta-1, TGF-beta-2, and TGF-beta-3) and xpected to accelerate reepithelialization and wound-healing y the activation of keratinocytes.10 Collagen type IV and aminin are mainly compositions of basement membrane and s pivotal for coherence between dermal layers and the epithe- ial. Our findings showed that repairing anal fistula with HAM esults in better outcome compared to simple repair. This is in oncordance with the results of other studies which reported he application of HAM in repairing recto-vaginal fistulas.6 We standardized histologic findings by using a modified coring system and provide a quantitative comparative con- ext. Although quantitative assessment of anal fistula healing rocess is challenging, we believe it would help researchers or more accurate comparison. Many surgical approaches for decrease healing time used uch as: Fistulotomy with 8.3% minor incontinence and 8.3% ecurrence rate,11 Advancement flap with 29% incontinence nd 10% recurrence,12 York Mason approach,13 Seton, Plug, brin glue14 or Stem cell injection with complex (high or trans- phincteric) anal fistulae. According to our knowledge, this study is first study to eval- ate the effect of HAM on wound healing post fistulotomy. he main positive point seems comparison of HAM effect by uantitative and qualitative measurement. 1 ;3 7(3):187–192 191 Conclusion Though the anal fistula is troublesome to the surgeons, it seems be improved by using the HAM graft. Our results seem to demonstrate that this technique is both simple and effective and would result in better surgical and histological outcomes comparing to simple repair. HAM increased rate of recovery and it suggested that HAM could be used for further research on patients’ treatment. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements This article was extracted from the thesis of Dr. Pirayeh, no. 5180, and approved by the research vice-chancellor of Shiraz University of Medical Sciences. Hereby, the authors would like to thank this vice-chancellery for financially supporting the study. e f e r e n c e s 1. Mushaya C, Bartlett L, Schulze B, Ho YH. Ligation of intersphincteric fistula tract compared with advancement flap for. Am J Surg. 2012;204:283–9. 2. Galis-Rozen E, Tulchinsky H, Rosen A, Eldar S, Rabau M, Stepanski A, et al. Long-term outcome of loose seton for complex anal fistula: a two-centre study of patients with and without Crohn’s disease. Colorectal Dis. 2010;12:358–62. 3. Rojanasakul A. LIFT procedure: a simplified technique for fistula-in-ano. Tech Coloproctol. 2009;13:237–40. 4. Meinero P, Mori L. Video-assisted anal fistula treatment (VAAFT): a novel sphincter-saving procedure for treating complex anal fistulas. Tech Coloproctol. 2011;15:417–22. 5. Jacob TJ, Perakath B, Keighley MR. Surgical intervention for anorectal fistula. Cochrane Database Syst Rev. 2010;12. CD006319. 6. Roshanravan R, Ghahramani L, Hosseinzadeh M, Mohammadipour M, Moslemi S, Rezaianzadeh A, et al. A new method to repair recto-vaginal fistula: Use of human amniotic membrane in an animal model. Adv Biomed Res. 2014;3:114. 7. Ahn JI, Lee DH, Ryu YH, Jang IK, Yoon MY, Shin YH, et al. Reconstruction of rabbit corneal epithelium on lyophilized amniotic membrane using the tilting dynamic culture method. Artificial Organs. 2007;31:711–21. 8. Ghahramani L, Jahromi AB, Dehghani MR, Ashraf MJ, Rahimikazerooni S, Rezaianzadeh A, et al. Evaluation of repair in duodenal perforation with human amniotic membrane: an animal model (dog). Adv Biomed Res. 2014;17:113. 9. Uludag M, Citgez B, Ozkaya O, Yetkin G, Ozcan O, Polat N, et al. Effects of amniotic membrane on the healing of primary colonic anastomoses in the cecal ligation and puncture model of secondary peritonitis in rats. Int J Colorectal Dis. 2009;24:559–67. 0. Loeffelbein DJ, Rohleder NH, Eddicks M, Baumann CM, Stoeckelhuber M, Wolff KD, et al. Evaluation of human amniotic membrane as a wound dressing for split-thickness skin-graft donor sites. BioMed Res Int. 2014:572183. 1. Pescatori M, Ayabaca SM, Cafaro D, Iannello A, Magrini S. Marsupialization of fistulotomy and fistulectomywounds http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0075 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http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0140 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0140 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0140 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0140 http://refhub.elsevier.com/S2237-9363(17)30045-X/sbref0140 A randomized trial study on the effect of amniotic membrane graft on wound healing process after anal fistulotomy Introduction Methodology Statistics Results Percent of healing Fecal incontinence parameter Pathology Discussion Conclusion Conflicts of interest Acknowledgements References