{"paper_id":"e47299a8-31b0-48ba-951a-c22c2c41b924","body_text":"Curcumin is Effective in Improving Folliculogenesis Profile \n(Primary and Secondary Folicles) and Oocytes Quality In \nVitro in Ectopic Endometriosis Mice (Mus Musculus) Models \n \nAulia Firmawati*,, and Widjiati \n \n1Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya \nUniver\nsity, Jl. MT Haryono Malang, Indonesia \n2Department of Embryology, Faculty of Veterinary Medicine, Airlangga University, Jl \nMulyorejo, Surabaya, Indonesia \n*e-mail: auliafirmawatidrh@gmail.com \n \n \nAbstract \nEndometriosis is the presence of ectopic endometrial tissue, such as that outside the uterine \ncavity, which can cause chronic inflammatory reactions. Curcumin is one of traditional herbal \nmedicines that is widely used. Some experiments have managed to find a mechanism to treat \nan ectopic endometriosis through the mechanism of suppression of several cytokines .This \nstudy was an experimental laboratory study, with four treatments and five replications, using \nfemale mice ( Mus musculus) that had reached puberty. The study c onsisted of three phases: \nthe first phase was the microscopic examination of in vitro oocytes quality, and the second \nwas histopathological examination of folliculogenesis profile using Hematoxillin eosin \nstaining. The results of this study showed signifi cant difference between positive control \ngroup (P0) with treatment and negative control (KN) groups in folliculogenesis profile (p \n<0.05). Examination of in vitro oocytes quality showed significant difference between \npositive control group with treatment g roup and negative control group (p <0.05). Curcumin \nhas several roles in cytokines modulation in ectopic endometriosis mouse models. Curcumin \nmay reduce the occurrence of apoptosis of granulosa cells so that it may directly improve \noocyte quality and folli culogenesis profile  (tersier follicel and de graf) . In conclusion, \ncurcumin effectively overcomes fertility problems through the mechanism of cytokines \nmodulation that plays a role in some cases of endometriosis such as: TNF -alpha, NF-kB and \nCOX-2. Curcumin may improve folliculogenesis profile and in vitro oocytes quality.\n \n \nKeywords: Ectopic endometriosis, Curcumin, Folliculogenesis profile, Oocyte quality \n \n \n1. INTRODUCTION \n \n Fertility in female animals is closely related to fertilization rate. Both depend on  the \nfolliculogenesis process and the quality of the ovum. It needs physiological coordination in \nthe ovary. Certain conditions may lead to a disruption in folliculogenesis, which may result in \ninfertility. One major cause of infertility is the ovulation f ailure. Other circumstances that \nmay be the most common cause of infertility is the formation of endometrial tissue on the \noutside of uterine cavity. This condition we commonly call with endometriosis (Herliana et \nal., 2008).  Endometriosis is a health prob lem that affects at least 10% in women of \nchildbearing age, 20 -50% in infertile women and may also occur in baboons ( Papio anubis) \nand also the long -tailed macaque ( Macaca fascicularis ) with a prevalence rate of 85%. \nEndomteriosis in baboons ( Papio anubis ) and the long -tailed macaque (Macaca fasiculata) \noccurrs because the two animals have common features with female humans in terms of the \n333\nCopyright © 2018, the Authors.  Published by Atlantis Press.\n \nThis is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).\n1st International Conference in One Health (ICOH 2017)\nAdvances in Health Sciences Research (AHSR), volume 5\n\n\nshape and pelvic anatomic shape and uterine type, reproductive and immunological \nphysiological characteristics (Grummer, 2006). In endometriosis model extracellular matrix \nbinding, which constitue bonds cumulus oophorus, is thickening (Herliana et al., 2008), and \ngranulosa cell may become apoptotic, which is also suspected as the cause of the decline in \noocyte quality. This adversely affects the fertility in the animal models. In addition, in the \ncase of endometriosis follicular period is extending, follicular growth is low and the size of \nthe dominant follicle in endometriotic women is decreasing (Gupta, 2008). \n Rats (Rattus norwegicus) and mice (Mus musculus) model of endometriosis show low \nfertility and the presence of nodules on pelvic region. Permana (2007) found that mice ( Mus \nmusculus) had decreased lymphocyte function, by mechanisms that are allegedly due to the \nsuppression of the proliferation and differentiation of lymphocytes, also a feature of \nangiogenesis in uterine area, ovaries and peritoneal front wall.  Today, herbal remedies \nincreasingly popular are increasingly popular, used also for alternative therapies in  the field \nof gynecological diseases, such as endometriosis and ovarian carcinoma. This is because \nherbal medicines do not have any significant side effects compared to synthetic drugs in the \nmarket during this time. One of the traditional herbal medicines \nwhich have been used to treat \nendometriosis is curcumin, an active ingredient extracted from Curcuma longa. Curcumin is a \nnatural yellow pigment of turmeric. Its content is approximately 3 -4% of turmeric. Curcumin \nhas also been shown to function as anti -inflammatory, anti -oxidant and anti -cancer. Some \nexperimental test has found the mechanism by which curcumin treats endometriosis, which is \nthrough the suppression of some of proinflammatory cytokines, such as TNF -alpha, barriers \nCOX-2 and NF-kappabeta (Wiesser et al., 2007).  In this study, we used experimental \nanimals SCID (Severe Combined Immunodeficient) mice ( Mus musculus) as an \nendometriotic model. Awwad et al., (1999) had successfully performed the implantation of \nendometriosis in mice ( Mus musculus) with a success rate of 96.5%. We, therefore, \ninvestigated the effectiveness of curcumin in endometriosis treatment. Until then, studies on \nthe effectiveness of curcumin in ectopic endometriosis mouse ( Mus musculus ) model on \nfertility have never been done. \n \n2.\n METHODS \n \n The study was conducted at in vitro fertilization laboratory and histopathological \nexamination was conducted at the Laboratory of Anatomic Pathology, Faculty of Veterinary \nMedicine, Universitas Airlangga, Surabaya. The study was performed for th ree months. \nExperimental animals were 2\n0 virgin female mice aged 3 months with an average weight of \n20-30 grams. Vasectomized male mice were as many as 20 mice and adult male mice were 4. \nMaterials used in this study were as follows: 0.9% physiological Sod ium Chloride (NaCl), \nDiionize Water (DW), Phosphate Buffer Saline (PBS), MEM, endometrial tissue of cysts \nmodel at Dr. Soetomo Hospital, Surabaya, penicillin, streptomycin, estradiol, curcumin, \ncyclosporin A and mineral oil, 70% alcohol, sterile paper tisu ue, and distilled water.  \nEquipments used in this study were as follows: Pasteur pipette, Eppendroff, disposable petri \ndish sized 30 x 80 mm, glass petri dish, 1 ml and 5 ml disposable syringes, laminar flow, \ninverted microscopes, centrifuges, Bunsen burner s, analytical balance, 5% CO2 incubator \nwith a temperature of 38.5 degrees C and a digital camera. \n The making of endometriotic mouse models.  First, the immune system of the mice \nwas suppressed using cyclosporine A. After reconstitution with water for inje ction, \ncyclosporine A was injected on day 1 intramuscularly in femoral biceps muscles at a dose of \n1.8 mg/mouse. Then, estrogen was injected intramuscularly in femoral biceps muscles on day \n1 and 5 after the injection of female treatment endometrium. There after, implantation of \nendometrial tissue was done on day -1 by injecting endometrial isolate intraperitoneally as \n334\nAdvances in Health Sciences Research (AHSR), volume 5\n\n\nmuch as 0.1 cc, equivalent to 0.6 grams of wet endometrial tissue (Awwad et al., 1999).  \nFolliculogenesis profile . Examination was done by taki ng the right and left ovary. \nFurthermore, both ovaries were made as histological preparations and primary and secondary \nfollicle counts in the ovaries were observed under a light microscope with a magnification of \n400 times. Each ovary was made into three preparations, the anterior, medial and a third \nposterior part. The result was the sum of total count observed in both ovaries.  Oocyte \nquality. Examination was done by collecting oocytes from fertilization pouch, then oocytes \nthat remained with Cumulus Ooph orus Complex (COC) was photographed using digital \ncamera Canon Ixus 95 I inverted microscope magnification 100x and camera magnification \n3,0x. Thereafter, COC circumference width was measured using the software Miotic Image II \nPlus. \n \n3.\n RESULTS AND DISCUSSION \n \nFolliculogenetic profile \n Determination of folliculogenesis development in the ovary of endometriotic mouse \n(Mus musculus) was done with a series of microscopic observation to count the number of \nprimary, secondary, tertiary and de Graaf's follicles. Obs ervation and calculation of follicle \ncount was based on the desired characteristics possessed by each follicle (Table 1). \n \nTable 1. Folliculogenesis profile. \nTreatment Folliculogenesis profile \nPrimary follicles Secondary follicles \nKN 17.00a ± 5.83 21.83ab  ± 4.44 \nP0 19.16a ± 4.21 30.83a  ±  8.01 \nP1 18.16a ± 6. 36 27.83a  ± 2.63 \nP2 16.33a ± 6.50 24.50ab  ± 7.20 \nNotes: a,ab,abc,c: different superscripts in the same column indicate the presence \nof a highly significant difference (p <0.05) with LSD \n \n The results showed that the group of ectopic endometriosis mice receiving placebo \n(P0) and the group receiving curcumin of 24 mg/kg bw/day (P1) had the highest mean \nprimary and secondary follicle count among the negative control group and other treatment \ngroups. It may result from extended periods of follicular period due to granulosa cell \napoptosis. This extension lead to follicular growth disorder characterized by oocyte follicle \ngrowth disorders, oocytes proliferation and differentiation. It indirectly resu lts in decreased \nFSH and GDF -9 levels. This condition leads to resisted expressing mechanisms of FSH \nreceptor, activin and GDF-9 produced by the granulosa cells, that are thought to play a role in \nstimulating FSH receptor expression through autocrine/paracrine mechanism. \n Decreased FSH and GDF -9 levels on granulosa cell due excessive apoptosis in  this \ngranulosa cells have caused disruption of enzymatic activity that is useful for catalyzing \nandrogens aromatization to produce estrogen, so it causes inhibite d steroidogenesis process \nand decreased estrogen sensitivity to provide feedback to the pituitary anterior so that GnRH \nmay produce LH. As a result, this will affect oocyte maturation process resulting, in extended \nfollicular periods that  results in folli culogenesis disturbance in the positive control group \n(P0). In treatment group 1 (P1) mean primary and secondary follicles counts were high. This \nmay be because the administration of curcumin doses in this group was less effective, so that \nthe process with in the inhibition of some cytokines such as TNF -alpha, COX-2 and NF -kB \nless than optimal in improving the condition of ectopic endometriosis. The primary follicle \nprofile can be seen in Figure 1. \n335\nAdvances in Health Sciences Research (AHSR), volume 5\n\n\n \nFigure 1. Folliculogenesis profile. Forms of primary follicles in each of the control and treatment \ngroups. Observation was made at 400x microscope magnification . Sign (    ) indicates the shape of \nprimary follicles. \n \nCurcumin is one of the herbal remedies that can suppress NF-kB and cytokines \ninflamatory pathway and also target genes of NF -kB cytokines in endometriotic models. \nWieser et al., (2007) demonstrated curcumin effects on endometriotic stromal cells. \nCurcumin inhibits the induction of pro -inflammatory cytokines, angiogenic cytokines and \nmacrophage migration inhibitory factor by NF -kB in in vitro model. Several recent studies \nalso found the modulatory effect of curcumin on several important molecular targets (TNF, \nIL-1, IL-6) and enzyme (COX -2) (Wieser et al., 2007). In addition, curcumin can also lower  \nanti-apoptotic genes expression, antioxidants and anti -angiogenesis effects (Sharma e t al., \n2005; Joe et al., 2008).  Inhibition of excessive TNF -alpha secretion and pro -inflammatory \ncytokines through suppression of NF -kB and NF -kB target gene cytokines pa thway by \ncurcumin in ectopic endometriosis mouse model of through modulation mechanism of \nseveral pro -inflammatory molecules targets in ectopic endometriosis mouse model, \nparticularly molecule targets TNF -alpha, enzyme cyclooxygenase -2 (COX -2) and xanthine  \noxidase, which can reduce the occurrence of apoptosis of granulosa cells, which may increase \nFSH level along GDF-9, which may enhance follicular development. The secondary follicles \nprofile can be seen in Figure 2. \n \n \n \n \n \n \n \n  \nFigure 2.  Folliculogenesis profile of secondary follicles in each control and treatment groups. \nObservations made at 400x microscope magnification. Sign (   ) indicates the shape of \nsecondary follicles. \n  \nIncreased FSH level on granulosa cells may enhance enzymatic activity useful fo r \ncatalyzing androgens aromatization or the like to produce estrogen. These activities are \nsuggested to be organized by an increase in adenylate cyclase and androgen action. Estrogen \n(estradiol), which is synthesized by the dominant follicle, also plays a role in increased FSH \nfollicular cells action to improve LH response. In addition to producing estrogen, FSH also \nplays a role in egg maturation, especially on the stages of follicular change. In addition, GDF-\n9 level in granulosa cells may increase so that GDF-9 is capable of running proliferation and \ndifferentiation of granulosa cells so well that the proliferation of theca cells will not be \ndisturbed. Interstitial theca cells have cell receptors for luteinizing hormone and insulin, in \nresponse to LH and insulin stimulation. The cells may produce high levels of androgens \n(Anwar, 2005). \n \n \n \n336\nAdvances in Health Sciences Research (AHSR), volume 5\n\n\nOocyte quality \nDetermining the quality of oocytes was done by observing the oocyte microscopically \nto observe cumulus ooporus circumference  (Table 2). Based on Analysis of Variant \n(ANOVA) followed by LSD test it has been found that the group endometriosic ectopic mice \nmodel receiving curcumin at 36 mg/kg/day (P2) were highly significantly (p <0.05) different \nfrom ectopic endometriosis mice model receiving curcumin at 24 mg/k g bw/day (P1) and \nmi\nce without treatment (KN). \n \nTable 2. Oocytes quality \nTreatment Oocytes count \nfrom IVF \nOocye quality observation \n \nKN 134 2,98b  ± 0,239 \nP0 0 0c ± 0 \nP1 94 3,07b ± 0,125 \nP2 124 3,60a  ± 0, 256 \nNotes: a, b, c : different superscript in the same column indicate the \npresence of a highly significant difference (p <0.05) with Tukey's \nHSD test. \n \nIt shows that curcumin administration to ectopic endometriosis mouse models can \nimprove oocytes quality (cumulus oophorus complex circumference) by inhibiting apoptosis \nof granulosa cells through modulation mechanism of several targets of pro-inflammatory \nmolecules ectopic endometriosis mouse models, especially TNF-alpha, enzyme \ncyclooxygenase-2 (COX-2) and xanthine oxidase so as to increase GDF-9 level and decrease \nhyaluronant level in ectopic endometriosis mouse models. Inhibition mechanism of molecule \ntarget in ectopic endometriosis mouse models with curcumin is expected to improve the \nproliferation and differentiation of granulosa cells, that can also improve cumulus expansion \nwhich may directly improve oocyte quality by increasing Cumulus Oophorus Complex \n(COC) circumference. \n \nREFERENCES \n1.\n Anwar, R. 2005. Morfologi dan Fungsi Ovarium. Subbagian Fertilitas dan \nEndokrinologi Reproduksi. Fakultas Kedokteran Unniversitas Padjajaran. Bandung. \n2. Awwad J.T., RA Sayegh, XJ Tao. The SCID mouse : an experimental model for \nendometriosis. Human Reproduction. 1999 ; 14 (12) : 3107 – 11 \n3. Grummer R .2006. Animal models in endometriosis research; human reproduction \nupdate vol 12, no 5 pp.641-649. \n4.\n Gupta,S. 2008. Pathophisiology, infertility of endometriosis. \nhttp://endometriosis.edu.html. \n337\nAdvances in Health Sciences Research (AHSR), volume 5","source_license":"CC0","license_restricted":false}