{"paper_id":"bfdf1cc6-b9da-4d61-b510-8a701d76075f","body_text":"R E S E A R C H Open Access\nA pilot study to search possible mechanisms of\nultralong gonadotropin-releasing hormone agonist\ntherapy in IVF-ET patients with endometriosis\nHiroshi Tamura 1†, Akihisa Takasaki 2†, Yasuhiko Nakamura 3, Fumitaka Numa 4 and Norihiro Sugino 1*\nAbstract\nBackground: Additional treatment with a gonadotropin-releasing hormone (GnRH) agonist (GnRHa) before IVF-ET\n(ultralong GnRHa therapy) has been reported to improve the outcome of IVF-ET in endometriosis patients. However,\nthe mechanism of ultralong GnRHa therapy is unclear. It is suggested that inflammatory cytokines and oxidative\nstress contribute to infertility in endometriosis patients. Therefore, in order to search a possible mechanism of\nultralong GnRHa therapy, we investigated the effect of ultralong GnRHa therapy on intrafollicular concentrations of\ntumor necrosis factor alpha (TNF α), oxidative stress markers, and antioxidants in patients with endometriosis.\nMethods: Twenty-three infertile women with Stage III or IV endometriosis were recruited for this study. Eleven\npatients received three courses of GnRHa (1.8 mg s.c. every 28 days), followed by a standard controlled ovarian\nhyperstimulation (COH) for IVF-ET (ultralong group). The other 12 patients received a standard COH with mid-luteal\nphase GnRHa down-regulation (control group). The numbers of matured follicles and retrieved oocytes, fertilization\nrates, implantation rates, clinical pregnancy rate, and intrafollicular concentrations of TNFα, 8-hydroxy-2’-deoxyguanosine\n(8-OHdG) and hexanoyl-lysine adduct (HEL) as oxidative stress markers, and melatonin and Cu,Zu-superoxide dismutase\n(Cu,Zn-SOD) as antioxidants were compared between the two groups.\nResults: The numbers of mature follicles and retrieved oocytes, and fertilization rates did not differ between the two\ngroups. Implantation rates and pregnancy rates tended to be higher in the ultralong group (21.4% and 27.3%,\nrespectively) compared with the control group (8.3% and 8.3%, respectively). TNFα concentrations in the follicular fluid\nwere significantly lower in the ultralong group (5.8 ± 3.2 pg/ml) than those in the control group (10.6 ± 3.2 pg/ml).\nFollicular concentrations of 8-OHdG concentrations were significantly lower in the ultralong group (5.7 ± 1.6 ng/ml) than\nthose in the control group (6.6 ± 1.5 ng/ml), while melatonin concentrations were significantly higher in the ultralong\ngroup (139 ± 46 pg/ml) compared with the control group (86 ± 27 pg/ml).\nConclusions:Ultralong GnRHa therapy reduces the detrimental effects of cytotoxic cytokines and oxidative stress in the\novary in patients with endometriosis.\nKeywords: Endometriosis, GnRH agonist, Cytokine, Oxidative stress, Melatonin\nBackground\nEndometriosis is considered to be the most intractable\ncause of female infertility [1,2]. Possible causes of the in-\nfertility include poor quality of oocytes and embryos [3],\nimpaired fertilization [4,5], and impaired implantation\n[6], each of which can be induced by inflammation and\noxidative stress in the pelvic cavity [7-9]. In fact, increased\nlevels of inflammatory cytokines such as interleukin-6\n(IL-6), interleukin-8 (IL-8), and tumor necrosis factor-\nalpha (TNF α) have been observed in the peritoneal fluid\nin patients with endometriosis [10]. TNF α, which is pro-\nduced by activated macrophages and NK cells, is a key\nmolecule in endometriosis. TNF α in the peritoneal cavity\nimpairs oocytes and embryos by its cytotoxicity [11,12].\nTNFα also increases prostaglandin production by endo-\nmetrial epithelial cells, which in turn initiates a surge of\n* Correspondence: sugino@yamaguchi-u.ac.jp\n†Equal contributors\n1Department of Obstetrics and Gynecology, Yamaguchi University Graduate\nSchool of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan\nFull list of author information is available at the end of the article\n© 2014 Tamura et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative\nCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and\nreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain\nDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,\nunless otherwise stated.\nTamura et al. Journal of Ovarian Research 2014, 7:100\nhttp://www.ovarianresearch.com/content/7/1/100\n\nother inflammatory cytokines in the endometrium [13,14].\nThis surge of inflammatory cytokines has been proposed\nas a cause of endometriosis-related implantation failure\n[13,14]. In addition, endometriosis patients showed high\nconcentrations of TNF α in ovarian follicular fluids,\nwhich is associated with poor oocyte quality or impaired\nfertilization [11].\nC h r o n i ci n f l a m m a t i o ni nt h ep e l v i cc a v i t yo fe n d o -\nmetriosis patients also induces oxidative stress in both\nthe pelvic cavity and reproductive organs. Increased\nand activated macrophages and polymorphonuclear\nleucocytes in the peritoneal fluid produce large amounts of\nreactive oxygen species (ROS) in patients with endometri-\nosis [15]. Oxidative stress levels in the ovarian follicular\nfluid are higher in patients with endometriosis than in other\ninfertility patients without endometriosis [16]. ROS produc-\ntion by endometrium is increased in endometriosis patients\n[17]. Oxidative stress causes detrimental effects on cells\nthrough lipid peroxidation, protein oxidation, and DNA\ndamage. Oxidative stress in the peritoneal cavity is one\nof the causes of endometriosis-associated infertility [8].\nOxidative stress reduces oocyte quality [18-20] and im-\npairs endometrial receptivity [21]. Together, these find-\nings strongly suggest that oxidative stress contributes to\ninfertility in endometriosis patients.\nThe pregnancy outcomes of endometriosis patients who\nundergo in vitro fertilization and embryo transfer (IVF-ET)\nare generally poor [22], and th ere are no effective treat-\nments for endometriosis-related infertility. Additional treat-\nment with a gonadotropin-releasing hormone (GnRH)\nagonist (GnRHa) before IVF-ET (ultralong GnRHa therapy)\nimproved the outcome of IVF-ET in endometriosis pa-\ntients, as shown by increased numbers of retrieved oocytes\nand transferred embryos, and higher implantation and\npregnancy rates [23-25]. However, the mechanism of\nultralong GnRHa therapy is unclear. Therefore, we investi-\ngated the concentrations of TNF α and oxidative stress in\novarian follicular fluids to see if they can shed light on the\nmechanism by which ultralong GnRHa therapy improves\nthe IVF-ET outcome in endometriosis patients.\nMaterials and methods\nPatients and clinical study\nThis study was reviewed and app roved by the Institutional\nReview Board of Yamaguchi University Graduate School of\nMedicine. Informed consent was obtained from all the\npatients in this study. Twenty-three infertile female pa-\ntients with endometriosis were recruited for this study.\nThe mean age ± S.D. of the patients was 34.0 ± 3.3 yr.\nwith a range of 28 –40 yr. Diagnosis of endometriosis\nwas confirmed by laparoscopy, laparotomy, or transvaginal\naspiration of the ovarian endometrial cyst. Severity of\nendometriosis was scored according to the four-stage clas-\nsification of the revised American Society for Reproductive\nMedicine (rASRM) score, and all patients had stage III or\nIV endometriosis. Patients were nonsmokers and free from\nmajor medical illness includinghypertension; all were inter-\nested in becoming pregnant. Patients were excluded if they\nhad myoma, adenomyosis, a congenital uterine anomaly,\nor if they used any kind of sex-steroidal agent including\nestrogens, progesterone, androgens, and OC.\nEleven patients received three courses of GnRHa\n(1.8 mg s.c. every 28 days) (buserelin acetate, Suprecur;\nMochida Pharmaceutical Co. Ltd., Tokyo, Japan), followed\nby a standard controlled ovarian hyperstimulation (COH)\nfor IVF-ET (ultralong group). Withdrawal bleeding was\ninduced using estrogen (Premarin: conjugated estrogens\ntablets, Pfizer Pharmaceutical Co. Ltd., Japan) and proges-\nterone (Duphaston: dydrogesterone tablets, Daiichi-Sankyo\nCo. Ltd., Japan) before COH. COH was initiated from the\n2nd day of the IVF-ET cycle by injection of 225 IU FSH\n(Folyrmon P; Fuji Pharmaceutical Co. Ltd., Tokyo, Japan)\nfor 3 days, followed by a daily injection of 150 IU HMG\n(HMG F; Fuji Pharmaceutical Co. Ltd., Tokyo, Japan). Nasal\nspray GnRHa (900 μg/day buserelin acetate, Suprecur;\nMochida Pharmaceutical) was also given from the 2nd\nday of the IVF-ET cycle to continuously suppress pituit-\nary gonadotropin secretion until the injection of HCG\n(HCG Mochida 10,000 IU; Mochida Pharmaceutical) for\novulation induction. Ultralong group included one case\nwith a male factor.\nTwelve patients received a standard COH with mid-\nluteal phase GnRHa down-reg ulation (control group).\nIn the control group, nasal spray GnRHa (900 μg/day)\nwas given from the mid-luteal phase in the previous\ncycle to the time of HCG injection for ovulation induction\nof the IVF-ET cycle. COH was given in a manner similar\nto the ultralong group described above.\nWhen leading follicles reached 18 mm or more, HCG\nwas injected for ovulation induction. Oocyte retrieval\nwas carried out 35 h after HCG injection. Each mature\nfollicle (more than 18 mm in diameter) was aspirated\nseparately and the follicular fluid containing the oocyte\nwas collected. Immediately after removal of the oocyte,\neach of the follicular fluids was centrifuged at 300 x g for\n15 min to remove cellular components. The supernatant\nfrom each follicle was mixed in each patient and was kept\nat –80C until assayed. The numbers of matured follicles,\nretrieved oocytes and fertilized oocytes, and fertilization\nrates, implantation rates, and clinical pregnancy rates\nwere compared between the two groups. Concentrations\nof TNF α, IL-6, and oxidative stress markers; 8-hydroxy-\n2’-deoxyguanosine (8-OHdG) as a marker of DNA\ndamage and hexanoyl-lysine adduct (HEL) as a marker\nof lipid peroxidation, and Cu,Zu-superoxide dismutase\n(Cu,Zn-SOD) and melatonin, as antioxidants, in follicu-\nlar fluids were measured using an ELISA kit or a radio-\nimmunoassay described below.\nTamura et al. Journal of Ovarian Research 2014, 7:100 Page 2 of 6\nhttp://www.ovarianresearch.com/content/7/1/100\n\nMeasurement of TNF α, IL-6, oxidative stress markers, and\nantioxidants in follicular fluids\nConcentrations of TNF α and IL-6 were measured using\na Human TNF α ELISA kit and Human IL-6 ELISA kit\n(Thermo Fisher Scientific Pierce Biotechnology, Rockford,\nUSA), respectively. Each sample of follicular fluid (50 μl)\nwas used for duplicate assay according to the assay proto-\ncol. The sensitivity of TNF α was 2 pg/ml, and the coeffi-\ncients of variation (CV) for intra- and inter-assay were 4.5%\nand 5.2%, respectively. The sensitivity of IL-6 was 1 pg/ml,\nand the CV for intra- and inter-assay were <10%.\n8-OHdG concentrations were measured using a New\n8-OHdG Check ELISA (Japan Institute for the Control\nof Aging, Nikken SEIL Co. Ltd., Shizuoka, Japan) as we\nreported previously [26,27]. Each sample of follicular\nfluid (50 μl) filtered using an ultrafilter (cut off molecular\nweight 10 kDa) was used for duplicate assay. The sensitiv-\nity of 8-OHdG was 0.5 ng/ml, and the CV for intra- and\ninter-assay were 5.5% and 6.1%, respectively.\nHEL concentrations were measured using an ELISA kit\n(Japan Institute for the Control of Aging) as we reported\npreviously [26,27]. Each sample of follicular fluid (50 μl)\nwas pretreated with chymotrypsin to perform proteolysis,\nand filtered using an ultrafilter (cut off molecular weight\n10 kDa) for duplicate assay. The minimal detectable con-\ncentration of HEL was estimated to be 2 nmol/L.\nCu,Zn-SOD concentrations were measured using a\nHuman Cu/Zn-superoxide dismutase ELISA kit (Northwest\nLife Science Specialties, LLC, USA) as we reported previ-\nously [26,27]. Each sample of follicular fluid (20 μl) was\nused for duplicate assay according to assay protocol. The\nsensitivity of Cu,Zn-SOD was 0.04 ng/ml, and the CV for\nintra- and inter-assay were 5.1% and 5.8%, respectively.\nIntrafollicular concentrations of melatonin were mea-\nsured by radioimmunoassay (RIA) as we reported previ-\nously [28]. Each sample of follicular fluid (500 μl) was\nused for duplicate assay. The sensitivity of the assay\nwas 4.2 pg/ml, and the CV for intra- and inter-assay\nwere 6.3% and 4.9%, respectively.\nStatistical analysis\nStatistical analysis was carried out with SPSS for Windows\n13.0. The Mann –Whitney U-test using the Bonferroni\ncorrection and Fisher’s test were employed as appropriate.\nCorrelations were analyzed using Spearman ’sr a n kc o r r e l -\nation coefficient. Differences were considered to be signifi-\ncant if P <0.05.\nResults\nThere was no significant difference in the mean age of\nthe patients between the two groups (Table 1). These\ntreatments resulted in the ultralong group receiving a\ngreater dose of gonadotropin and a longer duration of\novarian stimulation (Table 1). The numbers of mature\nfollicles and retrieved oocytes, and fertilization rates\nwere not significantly different between the two groups\n(Table 1). Embryo transfer was carried out in 8 of 12\ncases in the control group and in 8 of 11 cases in the\nultralong group (Table 1). The implantation rate and\npregnancy rate were higher in the ultralong group\n(21.4% and 27.3%, respectively) compared with the control\ngroup (8.3% and 8.3%, respectively), but the differences\nwere not significant (T able 1).\nTNFα concentrations in the follicular fluid were signifi-\ncantly lower in the ultralong group (5.8 ± 3.2 pg/ml) than in\nthe control group (10.6 ± 3.2 pg/ml) (Figure 1). IL-6 was\nnot detected in the follicular fluid in either group. 8-OHdG\nconcentrations were slightly but significantly lower in the\nultralong group (5.7 ± 1.6 ng/ml) than in the control group\n(6.6 ± 1.5 ng/ml), whereas the follicular HEL concentra-\ntions were not significantly different (Figure 2). Melatonin\nconcentrations were significantly higher in the ultra-\nlong group (139.2 ± 45.7 pg/ml) than in the control\ngroup (85.6 ± 27.4 pg/ml), while Cu,Zn-SOD concen-\ntrations were not significantly different between the\ntwo groups (Figure 3).\nThe correlations between TNF α,8 - O H d Ga n dm e l a -\ntonin concentrations in the follicle were analyzed\nusing Spearman ’s rank correlation coefficient. There were\nslight positive correlations between TNF α and 8-OHdG\nTable 1 Clinical characteristics and IVF-ET data\nControl\ngroup\nUltralong\ngroup P value\nNo. of patients 12 11\nAge (yrs) 34.5 ± 3.4 33.5 ± 3.3 0.45\nGonadotropin dose (IU) 1502 ± 377 2209 ± 849\na 0.037\nDuration of COH (days) 8.6 ± 1.9 11.5 ± 3.4 a 0.032\nEstradiol (pg/ml) 1995 ± 1054 1280 ± 892 0.098\nNo. of follicles ( ≧15 mm) 7.1 ± 2.7 6.6 ± 3.8 0.79\nNo. of mature follicles ( ≧18 mm) 3.0 ± 2.0 3.2 ± 2.8 0.79\nNo. of oocytes retrieved 5.0 ± 2.9 5.7 ± 4.1 0.70\nFertilization rate (%) 51.7 (31/60) 39.7 (25/63) 0.21\nCases of IVF/ICSI 11 / 1 8 / 3\nCases of embryo transfer 8 8 0.79\nNo. of embryos transferred 1.2 ± 0.8 1.3 ± 1.0 0.74\nCases of 2 –4 cell\nembryo/blastcyst (cryopreserved) 4 (1) / 4 (1) 6 (0) / 2 (0)\nImplantation rate (%) 8.3 (1/12) 21.4 (3/14) 0.35\nPregnancy rate (%) 8.3 (1/12) 27.3 (3/11) 0.23\nTwenty-three infertile women with Stage IIIor IV endometriosis were recruited for\nthis study. Eleven patients received three courses of GnRHa (1.8 mg s.c. every\n28 days), followed by a standard controlled ovarian hyperstimulation (COH) for\nIVF-ET (ultralong group). Twelve patients received a standard COH with mid-luteal\nphase GnRHa down-regulation (control group). Data are shown as the mean ± SD.\na; significant difference (Fisher’st e s to rt h eM a n n–Whitney U-test using the\nBonferroni correction).\nTamura et al. Journal of Ovarian Research 2014, 7:100 Page 3 of 6\nhttp://www.ovarianresearch.com/content/7/1/100\n\n(R = 0.1178, P = 0.5924), and negative correlations be-\ntween TNF α and melatonin (R = −0.0893, P = 0.6852)\nand melatonin and 8-OHdG (R = −0.3976, P = 0.0602).\nHowever, the trends were not statistically significant\nbecause of a small sample size.\nDiscussion\nThe present result clearly showed that the concentra-\ntions of a cytotoxic cytokine (TNF α) and oxidative stress\n(8-OHdG) in follicular fluids were significantly lower in\nthe ultralong GnRHa therapy group than in the control\ngroup, suggesting a potential mechanism that additional\nGnRHa treatment before IVF-ET improves the pregnancy\noutcome of IVF-ET by reducing the detrimental effects of\ncytotoxic cytokines and oxidative stress in the peritoneal\nenvironment or implantation environment in patients\nwith endometriosis. TNF α and oxidative stress in ovarian\nfollicles not only damage oocytes and embryos leading\nto the impaired fertilization, but also impair endometrial\nreceptivity leading to implantation failure in patients with\nendometriosis [10-14].\nAlthough ultralong GnRHa therapy reduced the con-\ncentrations of TNF α and oxidative stress markers in\novarian follicles, it is unclear whether it also reduced\nthem in the peritoneal cavity and in the endometrium.\nSince it is reported that the cytokine levels were de-\ncreased by GnRHa treatment in the peritoneal cavity as\nwell as in the ovary [29,30], there is a possibility that\nthe hostile environment of the peritoneal cavity and\nendometrial cavity was improved by ultralong GnRHa\ntherapy in this study. Therefore, we hypothesize that\nthe decrease in TNF α and oxidative stress by ultralong\nG n R H at h e r a p ym a yh a v ec o n t r i b u t e dt ot h ei m p r o v e -\nment of implantation rate and pregnancy rate.\nInterestingly, ultralong GnRHa therapy increased the\nmelatonin concentrations in the follicular fluid (Figure 3).\nMelatonin is a hormone secreted by the pineal gland,\nand regulates a variety of central and peripheral actions\nrelated to circadian rhythms and reproduction. Melatonin\nis a powerful free radical scavenger and a broad-spectrum\nantioxidant [31,32]. We previously demonstrated that\nmelatonin is present in human ovarian follicles and\nthat its concentration increases during follicular growth\n[18-20]. We also reported that melatonin is taken up\ninto the follicular fluid from the blood, and that it pro-\ntects oocytes from ROS within the follicle during ovulation\nFigure 1 Tumor necrosis factor alpha (TNF α) concentrations in\nfollicular fluids. Twenty-three infertile women with Stage III or IV\nendometriosis were recruited for this study. Eleven patients received three\ncourses of GnRHa (1.8 mg s.c. every 28 days), followed by a standard\ncontrolled ovarian hyperstimulation(COH) for IVF-ET (ultralong group).\nTwelve patients received a standard COH with mid-luteal phase GnRHa\ndown-regulation (control group). TNFα concentrations were measured in\nthe follicular fluid obtained at the time of oocyte retrieval. Values are\nmean ± SD. Statistical analysis was employed with the Mann–Whitney\nU-test using the Bonferroni correction.\nFigure 2 Concentrations of oxidative stress markers in follicular fluids. Twenty-three infertile women with Stage III or IV endometriosis were\nrecruited for this study. Eleven patients received three courses of GnRHa (1.8 mg s.c. every 28 days), followed by a standard controlled ovarian\nhyperstimulation (COH) for IVF-ET (ultralong group). Twelve patients received a standard COH with mid-luteal phase GnRHa down-regulation\n(control group). The levels of oxidative stress markers; 8-hydroxy-2 ’-deoxyguanosine (8-OHdG) as a marker of DNA damage and hexanoyl-lysine\nadduct (HEL) as a marker of lipid peroxidation, were measured in the follicular fluid obtained at the time of oocyte retrieval. Values are\nmean ± SD. Statistical analysis was employed with the Mann –Whitney U-test using the Bonferroni correction.\nTamura et al. Journal of Ovarian Research 2014, 7:100 Page 4 of 6\nhttp://www.ovarianresearch.com/content/7/1/100\n\n[18-20,33]. Reduced oxidative stress and increased anti-\noxidant activities by melatonin in follicular fluids by\nultralong GnRHa therapy may also have contributed to\nthe improvement of implantation rate and pregnancy\nrate. The mechanism by which ultralong GnRHa ther-\napy increases the melatonin concentration in the follicle\nis unclear. We speculate that ultralong GnRHa therapy\nm a yh a v ei m p r o v e dt h ef u n c t i o no ft h ef o l l i c l eb yr e d u -\ncing inflammation of the ovary so that the follicle can\neffectively take up melatonin.\nIt is unclear how TNF α, oxidative stress, and melatonin\ninteracts each other. There were slight positive correlations\nbetween TNFα and 8-OHdG, and negative correlations be-\ntween TNFα and melatonin, and melatonin and 8-OHdG,\nalthough the trends were not stat istically significant. These\nresults may suggest that TNF α induces oxidative stress\nand decreases melatonin levels in the follicle. In other\nwords, the reduced TNF α by ultralong GnRHa therapy\nmay be responsible for the decrease in oxidative stress and\nthe increase in melatonin in the follicle.\nOn the other hand, as a demerit of the ultralong\nGnRHa therapy, our results showed a need for greater\ngonadotropin doses and longer COH days than in\npatients who received a standard mid-luteal GnRHa\ndown-regulation protocol, which is also consistent\nwith previous reports [34].\nUnfortunately, the present study did not clearly show\nthat ultralong GnRHa therap y improves the fertility of\npatients with endometriosis. This may be due to the\nsmall sample size of this study. A large-scale randomized\ncontrolled trial will be necessary to evaluate the efficacy\nof ultralong GnRHa therapy on pregnancy outcome in\npatients with endometriosis. It is also interesting to in-\nvestigate whether there are any differences in follicular\nlevels of TNF α, 8-OHdG, and melatonin between the\nfollicles containing fertilized and unfertilized, implanted and\nnon-implanted, or pregnantand non-pregnant oocytes.\nConclusions\nThis study suggested a possible mechanism of ultralong\nGnRHa therapy to improve the pregnancy outcome of\nIVF-ET, which is the reduction of the detrimental ef-\nfect of cytokines and oxidat ive stress in the peritoneal\nenvironment or implantation environment in patients\nwith endometriosis.\nCompeting interests\nNS has received financial support for research from Mochida Pharmaceutical\nCo. Ltd., Tokyo, Japan, which is not directly related with this study.\nAuthors’ contributions\nHT and AT designed the study and wrote the manuscript. AT, YN and FN\ncollected and analysed the data. NS coordinated and supervised the study.\nAll authors read and approved the final manuscript.\nAuthor details\n1Department of Obstetrics and Gynecology, Yamaguchi University Graduate\nSchool of Medicine, Minamikogushi 1-1-1, Ube 755-8505, Japan. 2Department\nof Obstetrics and Gynecology, Saiseikai Shimonoseki General Hospital,\nYasuokacho 8-5-1, Shimonoseki 759-6603, Japan. 3Department of Obstetrics\nand Gynecology, Yamaguchi Grand Medical Center, Oazaosaki 77, Foufu\n747-8511, Japan. 4Department of Obstetrics and Gynecology, Tokuyama\nCentral Hospital, Koudacho 1-1, Syunan 745-8522, Japan.\nReceived: 26 August 2014 Accepted: 11 October 2014\nReferences\n1. Akande VA, Hunt LP, Cahill DJ, Jenkins JM: Differences in time to natural\nconception between women with unexplained infertility and infertile\nwomen with minor endometriosis. Hum Reprod 2004, 19:96–103.\n2. Collins JA, Burrows EA, Wilan AR: The prognosis for live birth among\nuntreated infertile couples. 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Kupker W, Schultze-Mosgau A, Diedrich K: Paracrine changes in the\nperitoneal environment of women with endometriosis. Hum Reprod\nUpdate 1998, 4:719–723.\n31. Reiter RJ, Tan DX, Fuentes-Broto L: Melatonin: a multitasking molecule.\nProg Brain Res 2010, 181:127–151.\n32. Tan DX, Manchester LC, Terron MP, Flores LJ, Reiter RJ: One molecule,\nmany derivatives: a never-ending interaction of melatonin with reactive\noxygen and nitrogen species? J Pineal Res 2007, 42:28–42.\n33. Tamura H, Takasaki A, Miwa I, Taniguchi K, Maekawa R, Asada H, Taketani T,\nMatsuoka A, Yamagata Y, Shimamura K, Morioka H, Ishikawa H, Reiter RJ,\nSugino N: Oxidative stress impairs oocyte quality and melatonin protects\noocytes from free radical damage and improves fertilization rate. J Pineal\nRes 2008, 44:280–287.\n34. Cota AM, Oliveira JB, Petersen CG, Mauri AL, Massaro FC, Silva LF, Nicoletti\nA, Cavagna M, Baruffi RL, Franco JG Jr: GnRH agonist versus GnRH\nantagonist in assisted reproduction cycles: oocyte morphology. Reprod\nBiol Endocrinol 2012, 10:33–40.\ndoi:10.1186/s13048-014-0100-8\nCite this article as: Tamura et al. : A pilot study to search possible\nmechanisms of ultralong gonadotropin-releasing hormone agonist therapy\nin IVF-ET patients with endometriosis. Journal of Ovarian Research\n2014 7:100.\nSubmit your next manuscript to BioMed Central\nand take full advantage of: \n• Convenient online submission\n• Thorough peer review\n• No space constraints or color figure charges\n• Immediate publication on acceptance\n• Inclusion in PubMed, CAS, Scopus and Google Scholar\n• Research which is freely available for redistribution\nSubmit your manuscript at \nwww.biomedcentral.com/submit\nTamura et al. Journal of Ovarian Research 2014, 7:100 Page 6 of 6\nhttp://www.ovarianresearch.com/content/7/1/100","source_license":"CC0","license_restricted":false}