{"paper_id":"aaf34b9f-79e1-4849-95c3-1fb8a7f8f78f","body_text":"Research Article\nClinical Obstetrics, Gynecology and Reproductive Medicine\nClin Obstet Gynecol Reprod Med, 2016        doi: 10.15761/COGRM.1000140  Volume 2(2): 157-160\nEffect of vaginal gestrinone in Pentravan® on \nendometriosis patients using Mirena®: A preliminary \nreport\nHugo Maia Jr.1*, Clarice Haddad1, Wilson SD dos Santos Junior2, Nathaniel Pinheiro3, Maria Cristina Hirsch4 and Anderson Silva Santo5 \n1Consultant physician and researcher, Instituto da Mulher, Itaigara Memorial Day Hospital, Salvador, Bahia, Brazil \n2Professor of Pharmacy, Universidade do Estado da Bahia (UNEB), Department of Life Sciences, Salvador, Bahia, Brazil \n3Pathologist, ImagePat, Pathology Institute, Salvador, Bahia Brazil\n4Chief Pharmacist, HTM Farmácia de Manipulação, Salvador, Bahia, Brazil\n5Undergraduate student of biomedicine, Mauricio Nassau University, Salvador, Bahia, Brazil\nAbstract\nObjective: To investigate the effect of low doses of vaginal gestrinone with oral Pinus pinaster extract and resveratrol on endometriosis-related pain in patients with \ndeep endometriosis using Mirena® and still experiencing symptoms during use of this levonorgestrel-releasing intrauterine system.\nPatient and methods: This was an open observational study conducted with 20 patients with deep endometriosis and severe pain to investigate the clinical effectiveness \nof low doses of vaginal gestrinone to improve pain and induce amenorrhea in patients not responding adequately to Mirena®.  In Group A (n=14), treatment with \nvaginal gestrinone, oral Pinus pinaster extract and resveratrol began 3-6 months after Mirena® insertion when it was found that these patients were not responding \nsatisfactorily to treatment. In Group B (n=6), patients initiated treatment with vaginal gestrinone, oral Pinus pinaster extract and resveratrol at the time of Mirena® \ninsertion. The effect of gestrinone on aromatase expression in the eutopic endometrium was also investigated by immunohistochemistry in all patients.\nResults: In Group A, the use of Mirena® alone resulted in a small but significant decrease in pain scores. However, these patients were still experiencing breakthrough \nbleeding and pelvic pain. The introduction of low doses of vaginal gestrinone in Pentravan® together with oral Pinus pinaster extract and resveratrol led to a further \ndecrease in pain score, rendering these patients pain-free by the end of the second month of this combination treatment. In Group B, pain scores similar to those \nfound in Group A after introduction of the combination treatment were achieved after the first treatment month.\nConclusion: Concomitant use of Mirena® with low doses of vaginal gestrinone in association with oral antioxidants is an effective treatment for deep endometriosis-\nrelated pain.\nCorrespondence to: Hugo Maia Jr., Instituto da Mulher, Itaigara Memorial Day \nHospital, Salvador, Bahia, Brazil, Tel: +55 71 3247 8216, E-mail: ceparh@uol.com.br\nKey words: endometriosis, Mirena®, Pentravan®, Pinus pinaster extract, resveratrol, \nvaginal gestrinone\nReceived: February 27, 2016; Accepted: April 09, 2016; Published: April 13, \n2016\nIntroduction \nInitial trials using a levonorgestrel intrauterine system (LNG-IUS) \nhave shown the  effectiveness of this device for the treatment of pelvic \npain and to reduce the size of lesions in deep infiltrating endometriosis \n[1,2]. However, despite these encouraging initial results, pain may \npersist in some patients [3]. This lack of response may be a consequence \nof the development of some form of progesterone resistance caused by a \nreduction in progesterone receptor isoform B in both the endometriosis \nlesions and the eutopic endometrium, triggered by exposure to \ninflammatory mediators [4,5]. One medical solution to this problem \nis to use the vaginal route to administer hormones that interact with the \nandrogen receptor instead of the progesterone receptor, combined with \nnatural NF-Kappa.b inhibitors to curb down inflammation. Absorption \nof the hormone through the vagina offers a unique advantage not offered \nby other routes of administration because of the first uterine pass effect, \nan effective mechanism through which to concentrate pharmacologically \nactive agents such as gestrinone in the pelvic organs before they are \ndiluted into the systemic circulation [6,7]. \nIn the present report, the effect of lower doses of vaginal gestrinone \nin Pentravan\n® together with a combination of natural polyphenols \nwith antioxidant and antiinflammatory properties was investigated in \npatients with deep endometriosis who continued to have breakthrough \nbleeding and pain despite being in use of Mirena\n®. Two polyphenols, \nPinus pinaster extract and resveratrol, were used concomitantly [8].  \nThese natural polyphenols are a complex mixture of flavonoids extracted \nfrom the bark of a pine tree (Pinus pinaster) or present in red wine \n(resveratrol), and are able to block both NF-Kappa.b and aromatase \nactivity [9,10]. The effect of vaginal gestrinone associated with these \nnatural polyphenols on aromatase expression in the endometrium of \nthese patients was also investigated.\nPatient and methods \nThis was an open clinical study to investigate the effect of \n\nMaia H (2016) Effect of vaginal gestrinone in Pentravan® on endometriosis patients using Mirena®:  A preliminary report\n Volume 2(2): 157-160Clin Obstet Gynecol Reprod Med, 2016        doi: 10.15761/COGRM.1000140\nan association of natural polyphenols and low doses of vaginal \ngestrinone on patients with endometriosis who were using Mirena ® \nbut who continued to experience pain and breakthrough bleeding. \nThe combined effects of vaginal gestrinone together with the natural \npolyphenols on pain scores and bleeding patterns were also evaluated \nin endometriosis patients in whom treatment was initiated at the time \nof Mirena\n® insertion.\nThe primary endpoint of this study was to investigate the combined \nuse of an intrauterine levonorgestrel system (IUS) (Mirena ®) with low \ndoses of vaginal gestrinone and natural polyphenols on pain scores \nand bleeding patterns in patients with deep endometriosis. Other \nassociated medical conditions such as ovarian endometriotic cysts \n(n=2), adenomyosis and uterine fibroids (n=12) were also present.\nThe patients formed two separate groups. Patients in Group \nA (n=14) had already been using Mirena\n® for 3-6 months prior to \ncommencing use of vaginal gestrinone (Fagron, the Netherlands) \ntogether with oral Pinus pinaster extract (Fagron, the Netherlands) and \nresveratrol (Fagron, the Netherlands). In Group B (n=6), patients with \ndeep endometriosis initiated treatment with gestrinone, oral Pinus \npinaster extract and resveratrol at the time of Mirena\n® insertion. Prior \nto initiating vaginal gestrinone treatment, all patients were evaluated \nclinically. Global pain scores, applied to both dysmenorrhea and \ndyspareunia, were rated by the patient at the time of the first interview \nprior to gestrinone use and 2 months after treatment using a visual \nanalogic scale in which 0 was indicative of no pain and 10 reflected \nthe worst pain imaginable. Pain scores prior to Mirena\n® insertion \nin Group A were obtained from the patients’ medical records and \nconfirmed retrospectively at the time of the first medical interview \nto initiate treatment with gestrinone, Pinus pinaster extract and \nresveratrol. In Group A, the patients using Mirena\n® were treated with a \ncombination of 2.5 mg of vaginal gestrinone in Pentravan® (Fagron, the \nNetherlands) twice a week, together with 100 mg of oral Pinus pinaster \nextract (Fagron, the Netherlands) and 30 mg of resveratrol (Fagron, \nthe Netherlands) daily. In Group B, these medications were initiated \nat the time of Mirena\n® insertion. In all cases, they were prepared by a \nlicensed compounding pharmacy. The Pinus pinaster extract (100 mg) \nand resveratrol (30 mg) were prepared and placed in the same capsule \nat a local compounding pharmacy supervised by the same pharmacists \n(MCH, WSDS). The Mirena\n® devices were manufactured by Bayer \nand were inserted by the same doctors (HM, CH) in all cases, under \nintracervical block achieved with 10 ml of lidocaine 1%. Gestrinone \nis licensed by the Brazilian drug regulatory authorities (ANVISA) for \nuse as an oral medication for the treatment of endometriosis. It was \nprepared for vaginal use at the concentration of 2.5 mg in Pentravan\n® \n(Fagron, the Netherlands), always by same pharmacist (WSDS).\nThe patients were instructed to insert the gestrinone/Pentravan ® \npreparation into the vagina at bedtime, twice a week, on Mondays and \nFridays, using a disposable plastic applicator. Resveratrol and Pinus \npinaster extract were dispensed together in the same V caps (Fagron, \nthe Netherlands) and patients were instructed to take one capsule \norally every day at bedtime. \nThis study was conducted at the Instituto da Mulher, Itaigara \nMemorial Hospital. The institution’s internal review board approved \nthe study protocol. The Instituto da Mulher is a private medical facility \nspecializing in women’s health and the treatment of endometriosis. All \npatients were counseled with respect to this treatment regimen and \ngave their informed consent to participate in the study. \nHysteroscopy with endometrial biopsy was performed in the \n14 patients with endometriosis in Group A prior to and after two \nmonths of gestrinone use to evaluate aromatase expression by \nimmunohistochemistry. During hysteroscopy, the uterine cavity was \nevaluated and an endometrial biopsy was taken, with samples being \nsent to pathology for routine histology and immunohistochemistry. \nThe same two surgeons (HM and CH) performed all the hysteroscopic \nprocedures. Anesthesia was achieved with the use of a paracervical \nblock and light intravenous sedation with propofol. When evaluation \nof the uterine cavity was complete, the hysteroscope was removed and \na 4 mm Karman curette attached to a 10 ml disposable plastic syringe \nwas introduced and the endometrium was aspirated. The samples \nwere immediately fixed in 4% formalin and sent to pathology. Routine \nhistology using hematoxylin & eosin (HE) staining was performed on \nall samples. The same pathologist (NP) performed both the routine \npathology and immunohistochemical evaluation of the endometrium. \nThe presence of aromatase expression in the endometrium was \ndetermined by immunohistochemistry following antigen retrieval. \nAromatase expression was investigated using a commercially available \nmonoclonal antibody supplied by Serotech, Raleigh, NC, USA. Antigen \nretrieval was performed using the Tris-EDTA buffer at pH 8.0. The \nreaction was revealed using the DAKO EnVision Flex detection system \n+ Linker followed by DAB + substrate chromogen mix (DAKO). The \npresence of aromatase expression was rated either as positive if there \nwas any detectable staining reaction in the endometrium or negative \nwhen no reaction was observed. \nStatistical analysis was performed using the chi-square test to \ndetect differences in the percentages of endometria testing positive for \naromatase expression before and after gestrinone use. Student’s t-test \nwas used to detect differences in mean pain scores before and after \ngestrinone treatment. Significance was established at p<0.05. \nResults \nThe effect of Gestrinone and Mirena ® on pain scores \nIn Group A (n=14), use of Mirena ® alone resulted in a modest \nbut significant reduction in pain score from a mean of 9 to a mean \nof 6 (p=0.01) after 3-6 months of use; however, the patients were \nstill reporting pain at this time and none were in amenorrhea. In all \ncases, the presence of breakthrough bleeding coincided with flare-ups \nof dysmenorrhea-like pain. Nevertheless, when gestrinone (2.5 mg) \ntwice weekly + daily oral Pinus pinaster extract and resveratrol were \nintroduced, dysmenorrhea pain scores further decreased after one \nmonth of treatment to a mean of 1 (p<0.0001). After the second month \nof this combination treatment, all patients in this group became pain-\nfree and amenorrheic. With respect to other forms of pelvic pain such \nas dyspareunia and dyschesia, pain scores decreased significantly from \na mean of 8 to a mean of 1 after the first month of gestrinone treatment. \nIn Group B, insertion of Mirena\n® with immediate implementation \nof a daily regimen of gestrinone + Pinus pinaster extract and resveratrol \nled to a significant reduction in dysmenorrhea scores from a mean of \n9 to a mean of 2 (p=0.004) after the first month of treatment. By the \nend of the second month of treatment, all patients were completely \npain-free and remained so throughout treatment. There were no \nstatistically significant differences in pain scores between Groups A \nand B at any time period following implementation of gestrinone with \nPinus pinaster extract and resveratrol. No untoward systemic effects \nwere found with these low doses of gestrinone except for acne in 20% \nof patients. \n\nMaia H (2016) Effect of vaginal gestrinone in Pentravan® on endometriosis patients using Mirena®:  A preliminary report\n Volume 2(2): 157-160Clin Obstet Gynecol Reprod Med, 2016        doi: 10.15761/COGRM.1000140\nAromatase expression in the endometrium \nIn endometriosis patients using Mirena ® and still experiencing \npain and breakthrough bleeding, aromatase expression was detected in \nthe endometrium of patients in 88% of the cases. Aromatase expression \nwas detected by immunohistochemistry solely in the stroma, while no \nstaining reaction was found in the glandular epithelium. In these cases, \nhistology revealed the presence of a strong decidual reaction in the \nendometrium. After the introduction of vaginal gestrinone with oral \nPinus pinaster extract and resveratrol, the percentage of aromatase-\npositive endometria decreased to 20% by the end of the second month \nof combination treatment. Aromatase expression remained positive \nin only three patients and these patients were still reporting pain, \nalbeit much less. The endometrium was labeled as basal/inactive and \nthe decidual reaction in the stroma disappeared. At hysteroscopy, the \nmucosa was thin and avascular.\nDiscussion\nThe present report showed that in patients with deep endometriosis \nexperiencing pain and breakthrough bleeding while in use of Mirena ® \nthe introduction of low doses of vaginal gestrinone in Pentravan ® \nwith oral Pinus pinaster extract and resveratrol was highly effective in \nimproving pain and inducing amenorrhea. \nIn patients with endometriosis, the use of Pinus pinaster extract \nwith resveratrol successfully potentiated the inhibitory effect of oral \ncontraceptives in extended regimens on aromatase expression in the \neutopic endometrium [8]. Since aromatase gene transcriptions are \ndirectly or indirectly increased by NF-kappa.b activation, the reduction \nin their expression in the eutopic endometrium of endometriosis \npatients following the use of gestrinone with Pinus pinaster extract + \nresveratrol may be a consequence of the more effective inhibition of \nthis transcription factor [11]. The unabated aromatase expression in \nthe eutopic endometrium of symptomatic endometriosis patients using \nMirena\n® suggests that the persistence of pain may be a consequence of \nthe continued expression of this enzyme in the endometrium, probably \ndue to the development of progesterone resistance caused by increased \ninflammation [4,5,11]. This is the most likely explanation for the lack \nof effect of the LNG-IUS on aromatase expression in the endometrium \nof these patients, since inhibition of this enzyme correlates positively \nwith higher rates of amenorrhea [8]. The persistence of pain in \nendometriosis patients using Mirena\n® may be caused by the presence of \ninflammatory cytokines and prostaglandins in the endometrium, since \nthese may reduce the expression of progesterone B receptors, rendering \nboth the endometrium and endometriosis lesions resistant to the effect \nof levonorgestrel [5,11,12]. In patients who experience breakthrough \nbleeding during continuous use of oral contraceptives containing \ngestodene/ethinylestradiol, NF-Kappa.b remains actively bound to cell \nnuclei where it will increase the transcription of several genes directly \nor indirectly related to inflammation [13]. This will eventually maintain \nthe transcription of the aromatase gene active and the ensuing local \nestrogen production will further augment inflammation [11]. Estrogens \ncan increase Cox-2 activity, thus increasing prostaglandin production, \nwhich in turn can stimulate the expression of the aromatase gene, thus \nestablishing a link between estrogens and increased inflammation \n[11,14,15]. The more effective suppression of aromatase expression \nfollowing vaginal gestrinone treatment with oral Pinus pinaster + \nresveratrol in endometriosis patients using Mirena\n® is a consequence \nof the reduction in inflammation prompted by activation of the \nandrogen receptor by gestrinone and the concomitant blockade of NF-\nKappa.b activity by the oral flavonoids. This results in higher rates of \namenorrhea and pain-free intervals after just one month of treatment. \nGestrinone acts in the endometrium by stimulating the androgen \nreceptors, and the ensuing inhibition of aromatase expression may \nbe a consequence of the reduction in inflammation, since gestrinone \nhas been shown to have no direct effect on aromatase expression in \ncultured cells from endometriotic cysts [16]. Inflammation plays a \npivotal role in the progression of endometriosis [11] and activation of \nthe androgen receptors may represent an alternative for those patients \nin whom progestin therapy is unsuccessful. The combination of \nMirena\n® with low doses of gestrinone and oral antioxidants effectively \ndiminished aromatase expression in the endometrium of patients in \nuse of Mirena\n® who had dysmenorrhea and breakthrough bleeding, \nthus rendering them pain-free and amenorrheic. Because of the \nstrong anti-progesterone and anti-estrogenic effect of gestrinone, the \ndecidual reaction in the stroma disappeared completely, rendering the \nendometrium thin and avascular. As shown in the present paper, the \ncombination of Mirena\n® with low doses of vaginal gestrinone was as \neffective as gestrinone used alone in higher doses, as shown previously \nby our group [7]. \nThe association of Mirena ® with vaginal gestrinone and oral \nantioxidants, as reported in the present paper, induces high rates of \namenorrhea, with a considerable improvement in pain after just one \nmonth of treatment, irrespective of whether this treatment begins at \nthe time of Mirena\n® insertion or afterwards in the case of patients who \nremain symptomatic after 3 to 6 months’ use of the device. The decrease \nin pain score was similar in the two groups. The use of low doses of \ngestrinone together with Mirena\n® may increase patient compliance by \nreducing the side effects and the cost of treatment, while maintaining \nthe same effectiveness as that achieved with higher doses. Since \nendometriosis is an inflammatory, estrogen-producing pathology, \nthe combination of the anti-estrogenic effects of gestrinone with the \nantioxidant properties of resveratrol and Pinus pinaster extract may \nimprove control of the disease, thus explaining the rapid relief of pelvic \npain experienced by these patients.\nReferences\n1. Fedele L, Bianchi S, Zanconato G, Portuese A, Raffaelli R (2001) Use of a levonorgestrel-\nreleasing intrauterine device in the treatment of rectovaginal endometriosis.  Fertil \nSteril 75: 485-488. [Crossref]\n2. Kruse C, Seyer-Hansen M, Forman A (2012) Diagnosis and treatment of rectovaginal \nendometriosis: an overview. Acta Obstet Gynecol Scand 91: 648-657. [Crossref]\n3. Ferrero S, Tramalloni D, Venturini PL, Remorgida V (2011) Vaginal danazol for \nwomen with rectovaginal endometriosis and pain symptoms persisting after insertion \nof a levonorgestrel-releasing intrauterine device. Int J Gynaecol Obstet 113: 116-119. \n[Crossref] \n4. 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[Crossref]\n15. Noble LS, Takayama K, Zeitoun KM, Putman JM, Johns DA, et al. (1997) Prostaglandin \nE2 stimulates aromatase expression in endometriosis-derived stromal cells.  J Clin \nEndocrinol Metab 82: 600-606. [Crossref]\n16. Wang Q, Zhao H, Xiang Q, Ju H, Han SM, et al. (2009) Effect of Yikun Neiyi Wan on \nthe expression of aromatase P450, COX-2, and ER related receptor in endometrial cells \nin vitro from patients with endometriosis. J Tradit Chin Med 29: 296-300. [Crossref]\nCopyright: ©2016 Maia H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, \ndistribution, and reproduction in any medium, provided the original author and source are credited.","source_license":"CC0","license_restricted":false}