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A randomized controlled study of the efficacy of misoprostol andhyaluronic acid in preventing adhesion formation after gynecologicalsurgery: a rat uterine horn model

Cihan Kaya a,*, Nurten Sever b, Huseyin Cengiz c, Sukru Yıldız c, Murat Ekin c, Levent Yasar c

a Van O  zalp State Hospital, Clinic of Obstetrics and Gynecology, Turkeyb Bakirkoy Dr Sadi Konuk Training and Research Hospital, Department of Pathology, Turkeyc Bakirkoy Dr Sadi Konuk Training and Research Hospital, Department of Obstetrics and Gynecology, Turkey

1. Introduction

Postoperative intra-abdominal adhesions occur in 50–95% of women who undergo gynecological surgery [1]. The developmentof peritoneal adhesions following abdominal and pelvic surgery

leads to clinical problems, including intestinal obstruction,infertility, and chronic pelvic pain   [2]. Adhesions remain a

potential problem for future surgical procedures, increasing healthexpenses and hospital readmission rates, and reducing the quality

of life of the patient   [3]. There are several described causes of adhesions, such as mechanical trauma, ischemia caused by suturesor electrocautery, presence of foreign bodies, tissue desiccation,

and infection [4]. Furthermore, during peritoneal healing followingsurgery, the increase in peritoneal fluid, cytokines, proteins and

fibrin formation can promote adhesion formation [5,6].Currently, there are various methods of preventing adhesion

formation such as the use of barrier materials, non-steroid anti-

inflammatory drugs, calcium channel blockers, corticosteroids,vitamin E, antihistamines, metformin, melatonin, progesterone,

estrogen, gonadotrophin-releasing hormone (GnRH)-agonists andantagonists, anticoagulants, fibrinolytic agents, and antibiotics [7–

10].Hyalobarrier1 gel is a highly viscous gel derived from

hyaluronan, obtained through an auto-crosslinking process, andused as an adhesion prevention agent in a variety of surgical

procedures [11–13]. Hyalobarrier1 gel can reduce the incidenceand severity of postoperative adhesions in cases of severe uterinedamage caused by laparotomy or laparoscopic myomectomies[14,15]. The gel can also be easily applied during laparoscopic and

hysteroscopic procedures.

European Journal of Obstetrics & Gynecology and Reproductive Biology 176 (2014) 44–49

A R T I C L E I N F O

 Article history:

Received 7 December 2013

Received in revised form 3 February 2014

Accepted 14 February 2014

Keywords:

Surgical adhesions

Hyaluronic acid

Misoprostol

PGE1

TGF b1

A B S T R A C T

Objective:   To investigate the effect of misoprostol in the reduction of adhesion formation aftergynecological surgery.Study design:  A double blind, randomized controlled experimental study was designed. Twenty-onefemale Wistar Hannover rats were divided into three groups as control, misoprostol and Hyalobarrier1

groups. A uterine horn adhesion model was created. After anesthesia induction, 1.5–2 cm injuries were

made to the each uterine horn by cautery. The control group received no special medications except for

the standard surgical procedure. The misoprostol group received 10 mcg/kg misoprostol in addition tothe standard surgical procedure, and the Hyalobarrier1 group received 1 cm3 ready-for-useHyalobarrier1 gel intraperitoneally in addition to the standard surgical procedure. After 14 days from

the first surgical procedure, adhesion scores were evaluated.Results:   The extent ( p < 0.001), severity ( p < 0.001), degree ( p < 0.001) and total adhesion score( p < 0.001) values of the control group were statistically higher than the values of misoprostol andHyalobarrier1 groups. The inflammation score value of misoprostol group was statistically lower than

control and Hyalobarrier1 groups ( p < 0.001).Conclusion:   In this study, we have found a newtherapeuticpotentialof misoprostol that maybe useful in

preventing pelvic adhesion and reducing inflammation scores.  2014 Elsevier Ireland Ltd. All rights reserved.

*   Corresponding author at: Van Ozalp State Hospital, Clinic of Obstetrics and

Gynecology, Cumhuriyet Mh., No. 3 Merkez Ozalp, Van, Turkey.Tel.: +90 212 414 73 72.

E-mail address:  drcihankaya@gmail.com (C. Kaya).

Contents lists available at  ScienceDirect

European Journal of Obstetrics & Gynecology andReproductive Biology

j o u r n a l h o m e p a g e :   w w w . e l s e v i e r . c o m / l o c a t e / e j o g r b

http://dx.doi.org/10.1016/j.ejogrb.2014.02.033

0301-2115/  2014 Elsevier Ireland Ltd. All rights reserved.

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Misoprostol, a synthetic analog of prostaglandin E1, is used as

an anti-ulcer drug that increases gastric mucus secretion andreduces gastric acid secretion [16]. Misoprostol is also used off-label by obstetricians for the induction of first trimester abortion[17]. Misoprostol hasbeen shown to alter enzymaticprocessesthat

cause degradation and remodeling of collagen fibers   [18].Nevertheless, no in vivo studies have demonstrated that miso-prostol can reduce adhesion formation. Misoprostol is commer-cially available as Cytotec1 (Ali Raif Ilac, Istanbul, Turkiye) in a

pharmaceutical complex with hydroxypropyl methylcellulose,microcrystalline cellulose, sodium starch glycolate, and hydroge-nated castor oil [19].

In this study, we aimed to investigate the effect of the

intraperitoneal use of misoprostol in the prevention of adhesionsby reducing collagen synthesis, which plays a major role in woundhealing and scar formation, compared with that of Hyalobarrier1

gel, which is known to prevent adhesions in clinical practice.

2. Materials and methods

The study was approved by the local ethical review board of the

Bagcilar Training and Research Hospital in Istanbul, Turkey, for theuse of laboratory animals, and was performed at the experimental

surgery training and education center of the same hospital(approval number: 2013–17).

 2.1. Animal maintenance and treatment 

Rats weighing approximately 180–260 g were housed under

the institutional review board’s guidelines for animal care using aday cycle of 14 h of light with free access to food and water. Apower analysis was performed to calculate the minimum sample

size required for the study (alpha error = 0.05 and 1-beta = 0.8) andat least 12 uterine horns were required for each study group. As10% of the animals are lost during the procedures, we included intotal 14 horns. The rats were randomly assigned to three study

groups, each consisting of seven rats, using computer-based

randomization. Twenty-one mature, nonpregnant, female WistarHannover rats (aged 8–10 weeks) were used as models for theexperimental induction of postoperative uterine horn adhesion, as

defined by Kelekci et al.   [20]. Animals were anesthetized using50 mg/kg of 10% ketamine hydrochloride (Ketasol; Richter PharmaAG, Wels, Austria) intramuscularly(IM) and 5 mg/kg of 2% xylazine(Rompun; Bayer Health Care LCC, Kansas, KS) IM. Prior to the

operation, the abdominal skin was shaved and disinfected using10% povidone-iodine solution (Batticon; Adeka Laboratories,Istanbul, Turkey). A 3-cm midline incision was made, and theuterine horns were exposed. Seven standard lesions were inflicted

in the antimesenteric surfaces of each uterine horn using a 10 Wbipolar cautery (Bovie1, FL, USA). An area of 1.5 cm  1.5 cmbetween the two main vascular branches of the lower side wall at

the level of each uterine horn was cauterized to produce anopposing lesion. The first group of rats served as the control groupandreceived no special medication. The second group (misoprostolgroup) received 10 mcg/kg misoprostol intraperitoneally (IP) usinga diluted Cytotec1 200 mcg tablet (Ali Raif Ilac, Istanbul, Turkiye).

The third group (Hyalobarrier1 gel group) received 1 mL of ready-to-use Hyalobarrier1 gel (Fidia Advanced Biopolymers S.r.I AbanoTerme, Italy) IP. The abdominal incision was closed in two layerswith a 4-0 polyglycolic acid suture (VicrylTM; Johnson and Johnson

Medical Limited, Ethicon Limited, UK) for the peritoneum and 3-0polyglactin suture for the skin.

After the animals recovered from surgery, they were housedseparately under controlled temperatures of 22 2   8C and a 14 h

light cycle with food and water ad libitum. The surgery waslimited to

approximately 15 min for each rat to prevent tissue drying at room

temperature.All thesurgicalprocedures were performed by thesame

researchers.

 2.2. Tissue sample collection and histopathology

Following the 14-day recovery period, the rats were sacrificedby cervical dislocation. The adhesion areas were evaluated andgraded macroscopically by two observers who were blinded to thestudy groups. The extent and severity of the adhesions were

evaluated using an established scoring system  [21]. The extent of adhesion spread to the uterine horn defect was scored as follows:0, no uterine adhesion; 1, 1–25% involvement, 2, 26–50%involvement, 3, 51–75% involvement, and 4, 76–100% involve-

ment. Adhesions were further characterized on gross examinationaccording to the severity of attachment between the uterine hornsand other abdominal organs as follows: 0, no adhesion, 1, filmyavascular, 2, vascular or opaque, and 3, cohesive. The degree of 

adhesion formation was evaluated and scored using the following;0, no adhesion; 1, the adhesion could be separated from the tissuewith gentle traction, 2, the adhesion could be separated from thetissue with moderate traction, 3, the adhesion could be separated

from the tissue by sharp dissection. The sum of the threeparameters was used as the total score for each group.

Tissue samples were obtained from all serosal surfaces whereadhesions had developed. One histologist evaluated all tissues and

was blinded to the origin of the samples. Tissues were graded forlevels of inflammation and fibrosis using previously publishedgrading scales. For histological analyses, the excised tissues werefixed in 10% buffered formalin solution for 24 h. After fixation, a

routine tissue-processing procedure was performed, and sampletissues were embedded in paraffin. Paraffin wax blocks were cutinto 4-mm thick sections. Prepared sections were then stained withhematoxylin and eosin, and levels of inflammation were scored as

follows: 0, no inflammation; 1, presence of giant cells, occasionallymphocytes, and plasma cells; 2, presence of giant cells, plasmacells, eosinophils, and neutrophils; and 3, presence of many

inflammatory cells and microabscesses. The level of fibrosis was

scored as follows: 0, no fibrosis; 1, minimal, loose; 2, moderate; 3,florid dense [22].

 2.3. Immunohistochemistry

Slides were immunostained with 100mg of transforminggrowth factor beta-1 (TGF-b1) rat polyclonal antibody (Acris

Antibodies GmbH; Schillerstraße 5, D-32052 Herford, Germany).The sections were incubated at 65   8C for 1 h overnight.

Deparaffinization was performed using the following series of washes: two xylene washes (3 min each), two 100% ethanol rinses

(3 min each), and one wash each of 95% ethanol, 70% ethanol, 50%ethanol, 30% ethanol, and Tris-buffered saline with 0.05% Tween(TBST; 3 min each wash on a shaker).

For antigen retrieval, 500 mL of distilled water was added to apressure cooker. The slides were immersed in a staining dishcontaining the antigen retrieval solution and then placed in a de-cloaking chamber. Sodium citrate buffer (10 mM Sodium citrate,0.05% Tween 20, pH 6.0) was used as the antigen retrieval buffer.

The sections were boiled for approximately 10 min and cooled atroom temperature. The slides were then removed and rinsed inTBST.

For the staining procedure, the slides were washed with TBST

and covered with 3% hydrogen peroxide to inactivate theendogenous peroxidase. The slides were washed three times withTBST for 3 min each on a shaker and blocked with a universalprotein blocking solution for 1 h. The TGF-b1 rat antibody

(AP06350PU-N) was diluted to 1:50. The primary antibody was

applied to each section and incubated overnight in a humidified

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chamber at 4   8C. The slides were washed three times with TBST

(3 min each on a shaker). Secondary HRP-conjugated antibodydiluted in the blocking solution was applied to each section as perthe manufacturer’s recommendations and incubated for 30 min at

room temperature. The slides were washed three times with TBST

(5 min each on a shaker) and incubated with freshly prepared 3,30-diaminobenzidine substrate until the stain developed. The sectionswere rinsed with water and counterstained with hematoxylin.

 Table 1

Comparison of clinical, histopathological and immunohystochemistry scores of the control, misoprostol and Hyalobarrier1 groups.

Parameters Control group Misoprostol group Hyalobarrier1 group   p-Value

Number of uterine horns 14 14 14

Clinical adhesion score

Extent score 2.29 0.73 [2 (1–3)]   0.860.86 [1 (0–2)]   0.860.86 [1 (0–2)]   0.0001Severity score 2.29 0.73 [2 (1–3)]   0.860.86 [1 (0–2)]   0.860.86 [1 (0–2)]   0.0001

Degree score 2.14 0.86 [2 (1–3)]   0.860.86 [1 (0–2)]   0.860.86 [1 (0–2)]   0.0001

Total score 6.712.13 [7 (3–9)]   2.572.59 [3 (0–6)]   2.572.47 [3 (0–6)]   0.0001

Histopathological scoreInflammation 2 0 [2 (2–2)]   1.430.51 [1 (1–2)]   2.140.36 [2 (2–3)]   0.0001

Fibrosis 1.29 0.47 [1 (1–2)]   1.430.51 [1 (1–2)]   1.290.47 [1 (1–2)]   0.658

Immunohystochemistry

TGF b1 antibody 1 0.78 [1 (0–2)]   0.710.47 [1 (0–1)]   0.860.66 [1 (0–2)]   0.581

TGF b1, transforming growth factor beta 1.

[

Fig. 1. Clinicaladhesion scoring.(a) No uterineadhesion, (b)1–25% involvement,filmy avascular, theadhesion could be separated fromtissuewith gentle traction, (c)26–50%

involvement,vascular and opaque, the adhesioncould be separated fromtissue withmoderate traction,and (d) 51–75% involvement,cohesive attachment of uterine hornto

each other or other abdominal organs, requiring sharp dissection.

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Samples were then dehydrated by two washes with 100% ethanol

(3 min each) and two rinses with xylene (3 min each). The slideswere mounted with cover slips and all sections examined using alight microscope. Five randomly chosen fields of each horn wereexamined, and the number of positively stained cells among

100 cells was counted. A total of 500 cells were counted for eachhorn. The percentage of immunoreactive cells was recorded by anestablished scoring systemas follows: 0, negative; 1,<33% positivestaining; 2, 33–66% positive staining; 3,   >66% positive staining

[23].

 2.4. Statistical analysis

Statistical data analysis was performed using the NCSS 2007Statistical Software (Number Cruncher Statistical System; NCSSLLC, Kaysville, UT 84037, USA). Descriptive statistical methods(mean, standard deviation, min–max measurements) were used

for data analysis. The Kruskal–Wallis test was used to comparedifferences in scores between the groups and the Dunn test wasused to compare differences between subgroups. A   p-value of 

<0.05 was considered statistically significant.

3. Results

 3.1. Macroscopic clinical adhesion scores

Total clinical adhesion formation scores were evaluated for thecontrol, misoprostol, and Hyalobarrier1 groups, and the scores

were 6.71 2.13, 2.57 2.59, and2.57 2.47 respectively (Table 1).

A statistically significant difference was found between the miso-

prostol and Hyalobarrier1 groupscomparedwith thecontrol group in

the total adhesion scores ( p = 0.0001), extent scores ( p = 0.0001), and

severity scores ( p = 0.0001). However, there was no significant

difference in these parameters between the misoprostol group and

the Hyalobarrier1 group. The scores for the degree of adhesion

formation were 2.14 0.86, 0.86 0.86, and 0.86 0.86 for the

control, misoprostol, and Hyalobarrier1 groups, respectively. A

statistically significant difference in these scores was noted between

themisoprostol andcontrol groups ( p = 0.002) and the Hyalobarrier1

and control groups ( p = 0.002), but there was no statistically

significant difference in this parameter between the misoprostol

and Hyalobarrier1 groups (Table 2 and Fig. 1A–D).

 3.2. Microscopic histological features

The median histological inflammation scores were 2 0,

1.43 0.51, and 2.14 0.36 for the control, misoprostol, and

Hyalobarrier1 groups, respectively (Table 1). The inflammation score

of the misoprostol group was significantly lower than that of the

control and Hyalobarrier1 groups ( p = 0.0001) (Table 2). However,

there was no significant difference in the inflammation scores

between the control and Hyalobarrier1 groups(Fig.2A–D). There was

also no significant difference in the fibrosis score and immunohis-

topathological scoring with TGF-b1 (Fig. 3A and B) between the study

groups.

 Table 2

Comparison of  p values for clinical adhesion scores and inflammation scores of the

control, misoprostol and Hyalobarrier1 groups.

Dunn test Control group/

misoprostol

group

Control group/

Hyalobarrier1

group

Control group/

Hyalobarrier1

group

Extent score 0.0001 0.0001 0.999

Severity score 0.0001 0.0001 0.999

Degree score 0.002 0.002 0.999

Total score 0.0001 0.0001 0.999Inflammation 0.001 0.150 0.001

[

Fig. 2. Histopathologicalinflammation evaluation of adhered tissues. Hematoxylin and eosin 200. (a) Giant cellformation, (b) microabscesses formation, (c) minimal, loose

fibrosis, and (d) moderate fibrosis.

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4. Comment

Development of postoperative adhesions remains a majorhealthcare problem causing relaparotomy complications, extend-ed operation time, additional blood loss, and unexpected visceral

damage   [24]. Adhesions occur after damage to the mesothelialcells on the serosal surface of tissues, followed by inflammation

with the release of cytokines, and the formation of a soft fibrin gelmatrix. If this softfibrin is not fractioned and fibrinolytic activity is

insufficient, dense fibrinous and vascular changes develop withintwo weeks   [5]. To minimize this undesirable fibrin formation,several approaches have been suggested. These include minimiz-ing the tissue injury, removing the fibrin, and inhibiting the

proliferation of fibroblasts at the injured site  [10]. A number of agents have been studied for this purpose, such as fibrinolyticagents to reduce the fibrinous mass, nonsteroidal anti-inflamma-tory drugs, corticosteroids, statins, metformin, methylene blue,

cyclooxygenase-2 inhibitors, PPARc and VEGF receptor inhibitors,anti-oxidants, aromatase inhibitors, GnRH-agonists, and humanamniotic membrane. There is, however, no optimal method for

adhesion prevention [5,10,20,25–29].

Hyaluronan (or hyaluronic acid) derivatives prevent adhesionsby reducing fibrin formation and promoting the production of peritoneal mesothelial cells   [11]. Hyalobarrier1 is a reported

adhesion-preventing agent [15]. Furthermore, a systematic reviewand meta-analysis reported that the application of the auto-crosslinked hyaluronan gel significantly reduced the incidence of intraperitoneal adhesions after laparoscopic myomectomy and

incidence of intrauterine adhesions after hysteroscopy   [11].Hyalobarrier1 gel is also useful in its simple, ready-to-use form.In a multicenter study, Hyalobarrier1 did not significantly prolongoperating time, and the mean application time of the product was

two minutes [30]. In ourinstitution, Hyalobarrier1 gel isused asanadhesion-preventing agent in routine surgical procedures.

Misoprostol, a drug that is commonly used by obstetricians for

the induction of labor and facilitation of cervical dilation beforeintrauterine procedures, alters the microstructural architectureand enzymatic processes of collagen [16]. In our study, we showedthat both misoprostol and Hyalobarrier1 significantly reduced theclinical adhesion score compared with that of the control group. To

the best of our knowledge, this is the first study evaluating theeffect of misoprostol on adhesion prevention. Fibrin formationfollowing the inflammatory process is believed to be the majorfactor in the development of surgical adhesions. This inflammatory

process begins after tissue damage and lasts throughout collagenfiber synthesis that enhances scar formation [10]. Although themechanism of action of misoprostol in reducing collagen fiberformation has not been completely described, we found that the

histopathological inflammation score of the misoprostol group

was significantly lower than that of the Hyalobarrier

1

and control

groups. We can speculate that misoprostol may prevent adhesion

by reducing the action of inflammatory cytokines involved in thefirst step of fibrin formation.

In our study, we also performed fibrosis scoring and TGF-b1 ratantibody immunohistochemistry scoring. TGF-b1 has a well-

described role in cell differentiation, in triggering angiogenesis,and as a fibroblast activator in the inflammatory response  [31].

However, there was no significant difference found in the TGF-b1antibody immunohistochemistry scores between the control and

treatment groups. We postulate that, as a limitation of our study,the recovery period following the initial surgery was 14 days,which is the minimum stabilization time limit for collagensynthesis. Collagen band formation typically begins 3–5 days

following trauma and increases until stabilization at approximate-ly day 14 [32]. Therefore, assessments should be performed after14 days to provide more accurate immunohistochemistry results.

Most adhesion-preventing agents are administered locally in

daily surgical practice [30]. In our study, the preferred route of administration was IP because surgeons generally prefer applyingagents via small instruments to prevent adhesion during

minimally invasive surgical techniques. We used 10 mcg/kg of a

Cytotec1 tablet, as this was the described minimal dose of intraperitoneal misoprostol to produce its gastroprotective effect[33].

The limitations of our study are that the required dose of misoprostol was obtained by dissolving a Cytotec1 200 mcg tabletin distilled water and that the effects of the tablet components onadhesion prevention or formation are unknown. The optimal dose

and administration route of misoprostol are also unknown;therefore, pharmacokinetic studies should be performed on theuse of misoprostol in preventing pelvic adhesions. Moreover, thecellular and molecular interactions of misoprostol in this situation

need to be investigated further.

Conflicts of interest statement

The authors have no conflicts of interest to declare.

 Acknowledgements

We are grateful to thank to Aysenur Turan for her technicalsupport and to Rana Konyalioglu for statistical analyses of the data.

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