{"paper_id":"df6b4dce-8c49-4721-9bcf-8587083cbbb8","body_text":"Iranian Journal of Reproductive Medicine Vol.5. No.3. pp:89-93, Summer  2007 \n \nThe effect of pentoxifylline on the growth of \nendometrial implants and leukocytes in rats. \n  \nAfsaneh Mohammadzadeh1 M.D., Mahnaz Heidari2 M.Sc., Haleh Soltan Ghoraiee3 M.D., Marefat \nGhaffari Novin1 Ph.D., Mahmood Jeddi-Tehrani4 Ph.D., Mohammad Mahdi Akhondi1 Ph.D., Hojjat \nZeraati5 Ph.D.,  Farzaneh Mohammadzadeh6 M.Sc.,   Pegah Ebadi1. B.Sc. \n \n1 Department of Reproductive Endocrinology and Embryology, Reproductive Biotechnology Research \nCenter, Avesina Research Institute, ACECR, Tehran, Iran. \n2 Departmet of Nanotechnology , Nanobiotechnology Research Center , Avesina Research Institute, \nACECR, Tehran, Iran. \n3 Department of Reproductive and Biotechnology  Genetic, Reproductive Biotechnology Research \nCenter,  Avesina Research Institute ,ACECR, Tehran, Iran. \n 4 Department of Monoclonal Antibody, Monoclonal Antibody Research Center , Avesina Research \nInstitute ,ACERCR, Tehran, Iran. \n5 Department of Epidemiology and Biostatistics, Tehran University of Medical Science,  Tehran, Iran. \n6  Anatomy Department, Iran University of Medical Science, Tehran, Iran. \n \nReceived: 7 January 2006; accepted: 11 July 2007 \nAbstract \nBackground: Immune system disturbances have an important role in endometriosis \nwhich may lead to infertility. It seems that inflammatory cytokines  specially tumor  \nnecrosis factor alpha (TNF- α) which were produced by activated macrophages have an \nimportant role in pathology of endometri osis.  Based on this theory, anti TNF- α  drugs \nlike pentoxifylline (PX) are suggested as new drugs for Endometriosis.             \nObjective: This experimental study has been done on female rats to determine the \neffect of PX on the endometrial implants and leukocytes in serum. \nMaterial and Methods:  In proestrous phase, one horn of rat’s bicorn uterus was \nremoved surgically and the endometrium was im planted to different places as follows: \nsubcutaneous, peritoneum and near ovaries. After two months observation, female rats \ndivided into two groups randomly. In treate d group (n=10) PX (5mg/kg twice a day) \nand in control group (n=10), normal saline (same dose) were injected subcutaneously. \nThen, via second laparotomy and in the same phase of the cycles, the size of implants \nand the amount of leukocytes in serum were measured. \nResults: In treated group compared with contro l, the size of implants was decreased \nsignificantly in right subc utaneous  (8.05mm vs 13.50mm) p<0.01, left subcutaneous \n(7.64 mm vs 14mm) p<0.01, right ovary ( 6.64 mm vs 15.22mm) p<0.001 and left ovary \n(7.18 mm vs 14.56 mm) p<0.005. In treated gr oup, the total leukocyte count (5259.54 ± \n178.78 vs 15833.33 ±  259.27) p<0.02 was decreased. The number of esterous cycle \nwas similar in both groups. \nConclusion: PX can reduce the size of endometrial implants as well as leukocyte count. \n \nKey Words: Endometriosis, Pentoxifylline, TNF-α, Anti TNF-α drugs, Infertility, Rat. \n \nIntroduction \n \n    Endometriosis is defined as ectopic growth of \nendometrial   stroma  and   glandular   tissues (1,2). \n \n Correspondence Author:  \nDr Afsaneh Mohammadzade, Avesina infertility center, \nNeyavaran street, next to shahrdari clinc, Tehran, Iran. \nEmail: af23mohammadzadeh@yahoo.com \nAlthough nearly three quarters of century have \nbeen passed since the initial description of \nendometriosis (3), our current understanding of the \netiology and pathphysiology of the disease has still \nbeen remained unclear (1-4). After the first \ndescription of the disease by Sampson in 1927 \nseveral different hypotheses have been suggested \nto explain the mechanisms for the development of \n\nMohammadzadeh et al \nIranian Journal of Reproductive Medicine Vol.5. No.3. Summer 2007 90\nendometriosis (4,5). However, Sampson’s theory \nof retrograde menstruation has gained most \nsupportive evidence (4, 5). Retrograde \nmenstruation is the reflux of menses through \nfallopian tubes toward the ectopic sites especially \nin peritoneal cavity. There are some reports about \nendometriosis in surgical incision in women who \nhave tolerated operation especially cesarean \nsection. It seems it is due to direct implantation of \nviable endometrial cells into incision (4,5). These \nfindings support Sampson's theory as well (4, 5). \nIn addition,changes in cell-mediated immunity \nrelated with endometriosis  have been reported in \nwomen, but the relevant data are inconsistent (2-5). \nA reduction in proliferation of peripheral blood \nlymphocytes in response to recognition of \nendometrial antigens and cells has also been \nreported (2-6). \n   It was shown that the ratio of T-helper to T-\nsuppressor cells is increased in the peripheral \nblood of the affected women; on the contrary other \nstudies have indicated no remarkable differences in \nperipheral lymphocytes profiles (4-7).On the other \nhand, peritoneal macrophages have revealed an \nincrease in total number, concentration, and \nactivation status (8). \n    Tumor necrosis factor-alpha (TNF- α) is \nregarded as one of the cytokines that has been \ngained recent attention in the pathphysiology of \nautoimmune diseases (9). TNF- α is a major \nproduct of activated macrophages. It can activate \nthe inflammatory leukocytes which can lead to the \nproduction of other pro inflammatory cytokines, \nincluding interleukin-1 (IL-1), IL-6, and additional \nTNF-α (9). TNF- α can stimulate endometrial cell \nadhesion, as well as, induce matrix \nmetalloproteinases (MMPs) expression, both of \nwhich are necessary events in the initial \ndevelopment of endometriosis (10, 11). \n    Considering above mention facts, TNF- α has an \nimportant role in endometriosis, therefore anti \nTNF-α drugs may be proper drug for this disease. \nPX reduces both the production and action of \ncytokines such as TNF- α through the elevation of \nintracellular cyclic AMP levels and subsequent \ndown regulation of cytoki ne production and action \n(4, 5, 12).  \n    PX is a member of Methylgezantine family \nwhich inhibits Phosphodiesterase in platlets and \nleads to the elevation of CAMP levels in them and \ndecreases the aggregation of them in circulation. \nTherefore, it is useful in cerebrovascular diseases \n(4, 5,13). PX can inhibit phosphodiesterase in \nmonocytes and macrophages and suppress \ncytokineproduction specially TNF- α  i n  \nmacrophages (4,5,12,13).   \n    In other immunological disease like sever \nrefractory rheumatoid arthritis (14), inflammatory \nbowel disease (15), and recurrent aphthous \nstomatitis (16), PX had a suppressive  effect on \nthese diseases .Therefore , it seems that anti TNF-\nα effect of this drug can be useful in treatment of \nendometriosis. Based on several studies, rats are \ngood model for the study on endometriosis.  They \ndon’t have menstrual cy cles and endometriosis \nisn't induced in them spontaneously (17,18). \n    Steinleitner et al (1991) induced endometriosis \nin female rats surgically  and mentioned PX could \nincrease fertility rates in comparison with normal \nsaline (90% vs 2.3 %). They suggested that PX \ncould raise fertility rates in endometriosis in rats \n(19). Nothnick et al (1994) reported that PX could \nmodulate rat endometriotic  implant growth and \nproduce implant- specific proteins (ENDO-1& \nENDO-2) (20).  \n     Balach et al (1997) reported that PX versus \nplacebo could improve fertility rates in women but \nnot significantly (21). Although in animal studies \n(19, 20), PX could have a good effect on \nendometriosis, but the human studies couldn’t be \nable to show the significant changes in treated \ngroup with PX (21, 22).    \n     In order to determine the effect of PX on \nendometriosis, we decided to do our study in \nfemale rats. In this study, we induced \nendometriosis in female rats surgically and \nevaluated the effect of PX versus placebo on the \nendometriosis. \n    The aims of this study were investigating : 1) \nThe effect of PX on the growth of endometrial \nimplants in female rats especially in different \nplaces. 2)The effect of PX on leukocyte count in \nserum, in order to determine the immune response \nwith this drug. \n \nMaterials and methods \n \n     Our randomized clinical trial study was done on \n20 female rats’ sparauge- dwley (2 months old). \nThis study was performed in Avesina Research \nInstitute under support of Iranian Academic Center \nfor Education, Culture and Research (ACECR). \nThe rats were housed individually in hanging \ncages. The facility was maintained on a 14-h light, \n10-h darkness schedule. Female rats have 4- 5 days \nestrous cycles (24).  For evaluation of phases in \nfemale rats we did vagi nal Pap smear daily (25). \nOnly those rats exhibited normal 4-5 day estrous \ncycle were subjected to surgical induction of \n\nEffect of pentoxifylline on the growth of endometrial implants \n \nIranian Journal of Reproductive Medicine Vol.5. No.3. Summer 2007 91\nendometriosis. All of the ra ts had a file that the \nprocesses were written in them step by step. \n    In proestrous phase or the phase of follicular \ngrowth and peak estrogen secretion, rats were \nanesthetized intra peritoneally (i.p) with 20 mg/ kg \nketamine hydrochloride. In first operation, the left \nuterine horn and associated fat tissue were \nremoved. Left ovary was saved. The removed \nuterine horn was cut longitudely and divided to 6 \npieces (2×2mm). Then, endometrium was \nseparated from underling myometrium and 4 \npieces were sutured with 4-0 round nylon to the \ndifferent parts of peritoneal cavity; 1 piece near \nright kidney as right peritoneal implant, 1 piece \nnear mesanteric artery as left peritoneal implant \nand 2 pieces in both ovaries (25). Then, 2 pieces of \nendometrial implants were sutured in subcutaneous \nof the rat’s skin as right and left subcutaneous \nimplants (25).  \n    One hour after surgery, the animals could move \nand after 4 hours, they fed regularly. Two days \nlater, they were put near the male rat for starting \ntheir Estrous cycles (24,25). They were kept into \nclose consideration for 2 months. During this \nperiod, 10-12 Esterous cycles were recorded for \neach rat. Vaginal cytology was evaluated daily as \nan indirect index of ovarian activity and \nfortunately all of them had this normal Estrous \ncycle. \n    Rats (n=20) were randomly assigned to two \ngroups; in treated group (n=10), PX (manufactured \nby STADA, Ampoule, 100mg/5ml) in dose of \n5mg/kg twice a day was administered \nsubcutaneously (s.c) and in control group (n=10) \nnormal saline as placebo was administered with the \nsame dose (19, 20).  \nIn second laparotomy, which was done in \nProestrous phase too, at first we collected 5 \nmilliliter of blood by heart puncture and sent to \nlaboratory for measuring the number of Leukocyte \ncount  in serum. Then, the skin was cut and the \nendometrial tissues removed and the abdominal \ncavity was examined and all of our observations \nabout adhesions, cystic form ation, size and site of \nimplantation were recorded carefully. The \nendometriotic implants in each animal were \nmeasured (length x width) and the average size \n(mm\n2) for each place was calculated along with the \ntotal implant mass (mm2; defined as the sum of the \nsize of all implants in the same places) (20). The \ntissues were put in 10% formalin and sent to \nlaboratory for microscopic evaluation. \nEndometriosis was confirmed by observation of \ngland and stroma of endometrium in implants. The \nsurgeries were done by one surgeon and the slides \nwere evaluated by one pathologist blindly. \n \nStatistical analysis \n    These data were analyzed by SPSS program (t-\ntest and Mann-Whitney for quantitive and Chi-\nSquare and if needed Fisher exact test for qualitive \nvariants). p<0.05 was considered as significant. \n \nResults \n \n    Age, weight and size of implanted fragments \nwere similar in both groups. Daily pap smear \nshowed that the number of Esterous cycles was \nsimilar in both groups.  \n     The mean size of the implants in both groups is \nshown in Table I. In treated group, the size of \nimplants in right subcutaneous (8.05 vs. 13.50mm) \np<0.01, left subcutaneous (7.64 vs. 14.00 mm) \np<0.01, right ovary (6.64 vs. 15.22mm) p<0.001 \nand left ovary (7.18 vs. 14.56 mm)p<0.005 were \nsignificantly decreased. In right and left \nperitoneum (8.59 vs. 12.83 and 8.59 vs. 12.83 mm) \np<0.09, the size of implants were decreased in \ntreated group compare with control group but this \nwas not significant.\n \n    In treated group the results indicate, the total \ncount of Leukocyte was less than control group  \n(5259.54± 178.78 vs. 15833 ± 259.27) p<0.02 \n(Table II). The number of Estorous cycle was \nsimilar between two groups. \n \nTable I. Comparison between the size of endometrial implants in both group, based on the site of implantation. \n \nMean size(mm) in two groups  \nSite \nControl (n=10) Treated (n=10) \n \nP \nRight subcutaneous 13.50 8.05 0.01 \nLeft subcutaneous 14.00 7.64 0.01 \nRight peritoneum 12.83 8.59 0.09 \nLeft peritoneum 12.83 8.59 0.096 \nRight ovary 15.22 6.64 0.001 \nLeft ovary 14.56 7.18 0.005 \n\nMohammadzadeh et al \nIranian Journal of Reproductive Medicine Vol.5. No.3. Summer 2007 92\nTable II. Comparison of pathological reports between two groups, based on the site of implantation.   \nPathological report in two groups \n \n \nSite \nControl (n=10) Treated (n=10) \n \np \nRight subcutaneous 0- / 10+                             6- / 4+ 0.003 \nLeft subcutaneous 0- / 10+ 4- / 6+ 0.01 \nRight peritoneum 6- / 4+ 7- / 3+ 0.5 \nLeft Peritoneum 6- / 4+ 6- / 4+ 0.5 \nRight Ovary 1- / 8+                              8- / 2+ 0.03 \nLeft Ovary          3- / 8+ 7- / 2+ 0.00 \n- = No sign of endometriosis (stroma and gland of endometrium) in pathological report \n+ = There are sign of endometriosis ( stroma and gland of endometrium )in pathological report \n \n \nDiscussion \n \n    Our findings showed that in treated group with \nPX, the size of endometrial  implants were smaller \nthan control group .This finding was similar to \nNothnick et al study (1994) who reported that PX \ncould modulate growth in endometriotic implant \nrat and also rise in production process of implant- \nspecific proteins (ENDO-1& ENDO-2) (20).This is \ndifferent from our study because we studied the \nsize of endometrial fragments in different places \nand compared it with the same places in control \ngroup. \n    In treated group, the size of implants was \ndecreased significantly as follows: in right \nsubcutaneous, left subcut aneous, right ovary and \nleft ovary. In right and left peritoneum, the size of \nimplants was decreased in comparison with control \ngroup but not significantly. These findings suggest \nthat the suppressive effect of PX was not similar in \nall places.  This might be valuable when we are \nmaking decision for treatment of different types of \nendometriosis. \n     At the time of our study, there were a few \nstudies on the effect of PX in female rats (19, 20) \nand in human (21, 22). \n    Although in animal studies (19, 20), \nPentoxifylline could have a good effect on \nendometriosis, the human studies (21, 22) couldn’t \nbe able to show the significant changes in treated \ngroup with PX.    \n    In treated group, the total count of Leukocytes \nwas decreased. These findings suggested that \nendometriosis in rats was accompanied with \nimportant changes in\n Leukocyte count in serum \nand PX could decrease the total count of \nLeukocyte. It seems that alternation in immune \nsystem in endometriosis which might be \naccompanied with alternation in\n Leukocyte count, \ncan suppress endometrial growth in rats.  This \neffect of PX was not evaluated in different studies  \n \n \non rats (19-21). Therefore this finding is nearly \nnew and needs further studies for better evaluation. \nIn order to better understanding of the suppressive \neffect of the PX, we suggest further studies on \nnatural killer (NK) cells and TNF- α during \ntreatment. \nIn our study, PX didn’t have any adverse effect on \nmenstrual cycles in treated group. This is an \nimportant benefit in treatment with PX. Other \ndrugs suppress ovarian function and induce \nhypoestrogenic state in women. Hot flash and \nosteoporesis with these drugs can hurt women \n(4,5).  Based on our findings, under PX treatment, \novaries can secret hormones, these symptoms \naren’t seen and fertility ability will be intact. These \nfindings are similar to Steinleitner et al study \n(1991) findings. They induced endometriosis in \nfemale rats through surgical process and noticed \nthat treatment with intra peritoneal PX in \ncomparison with normal saline could increase \nfertility rates (90% vs. 2.3 %). They suggested that \nPX could increase fertility rates in endometriosis in \nrats (19).  \n    In this study we didn’t allow the rats to get \npregnant. This effect can be evaluated with future \nstudy. \n   \nConclusion \n \n    PX can decrease the size of endometrial \nimplants especially in ovaries and subcutaneous \nareas. In addition, significant reduction in \nLeukocyte count was noticed after PX therapy. \n \nReferences \n \n1. Akanda VA, Hunt LP, Cahill DJ, Jenkins JM.  \nDifferences in time to natural conception between \nwomen   with unexplained infertility and infertile \nwomen with minor endometriosis. 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